Patent Publication Number: US-2010129406-A1

Title: Holin-enhanced vaccines and reagents, and methods of use thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the priority benefit of U.S. Provisional Application No. 60/841,705, filed Sep. 1, 2006, the contents of which are hereby incorporated by reference herein in their entirety. 
    
    
     FIELD OF THE INVENTION 
     This invention relates generally to  Listeria  that express holin proteins and are useful for the delivery of heterologous polynucleotides and/or polypeptides. In particular, the  Listeria  are useful for delivery of heterologous polynucleotides and/or polypeptides to the cytosol of infected cells and in vaccines. 
     BACKGROUND OF THE INVENTION 
     Cancers and infections can be treated by administering reagents that modulate the immune system. These reagents include vaccines, cytokines, antibodies, and small molecules, such as CpG oligodeoxynucleotides and imidazoquinolines. Vaccines, including classical vaccines (inactivated whole organisms, extracts, or antigens), dendritic cell (DC) vaccines, and nucleic acid based vaccines, are all being applied to the treatment of cancers and infections (see, e.g., Robinson and Amara (2005) Nat. Med. Suppl. 11:S25-S32; Plotkin (2005) Nat. Med. Suppl. 11:S5-S11; Pashine, et al. (2005) Nat. Med. Suppl. 11:S63-S68; Larche and Wraith (2005) Nat. Med. Suppl. 11:S69-S76). 
     Another reagent of use in modulating the immune system is  Listeria monocytogenes  ( L. monocytogenes;  Lm).  L. monocytogenes  is an intracellular bacterium. Once the  Listeria  enters a host cell, the life cycle of the  Listeria  involves escape from the phagolysosome to the cytosol.  L. monocytogenes &#39; escape from the phagolysosome is mediated by listerial proteins, such as listeriolysin (LLO), PI PLC, and PC PLC (Portnoy, et al. (2002) J. Cell Biol. 158:409-414). The use of this reagent has been reported for the treatment of cancers and tumors (see, e.g., Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:13832-13837; Brockstedt, et al (2005) Nature Med. 11:853-860); Starks, et al. (2004) J. Immunol. 173:420-427; Shen, et al. (1995) Proc. Natl. Acad. Sci. USA 92:3987-3991).  Listeria -based vaccines are also reported, e.g., in U.S. Patent Publication Nos. 2005/0281783, 2005/0249748, 2004/0228877, and 2004/0197343, each of which is incorporated by reference herein in its entirety. 
     Improvements in the methods for  Listeria -mediated delivery of heterologous antigens to the cytosol of infected cells, especially antigen-presenting cells, are desired for the development of vaccines of increased efficacy. 
     SUMMARY OF THE INVENTION 
     The invention provides  Listeria  that are modified to express holin proteins and that are useful as heterologous antigen delivery vectors. In some embodiments, the delivery of heterologous polypeptides and/or polynucleotides from the  Listeria  to the cytosol of infected cells is enhanced or mediated by the holin proteins. Compositions such as pharmaceutical compositions and vaccines comprising the  Listeria  are provided. Methods of using the  Listeria  to induce immune responses or treat or prevent disease in mammals are further provided. Polynucleotides useful in the construction of the modified  Listeria  are also provided. 
     In one aspect, the invention provides a  Listeria  bacterium (e.g.,  Listeria monocytogenes ) comprising a first polynucleotide comprising a polynucleotide encoding a holin protein. In some embodiments, the bacterium further comprises a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein. In some embodiments, the bacterium further comprises a second polynucleotide comprising a polynucleotide encoding a heterologous polypeptide (e.g., a heterologous polypeptide comprising an antigen). The second polynucleotide may further comprise a promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide. In some embodiments, the bacterium may instead comprise a second polynucleotide comprising a polynucleotide encoding a self-replicating RNA which comprises a polynucleotide encoding a heterologous polypeptide. The second polynucleotide may, in some embodiments, further comprise a promoter that is operably linked to the polynucleotide encoding the self-replicating RNA. In some embodiments, the bacterium expresses the holin protein. In some embodiments, when the bacterium expresses the holin protein, the bacterium remains viable. In some embodiments, when the bacterium expresses the holin protein, the holin protein is expressed at at a level that does not substantially impair the growth of the bacterium and/or that does not lyse the cell membrane of the bacteria. In some embodiments, the holin protein is derived from a non-listerial bacterium or from a bacteriophage that is not a listeriophage. In some embodiments, the bacteria comprises: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide Populations comprising the  Listeria  are also provided. Pharmaceutical compositions, immunogenic compositions, and vaccines comprising the  Listeria  are further provided. In addition, methods of using the  Listeria  to induce an immune response to an antigen in a mammal, to treat a disease (e.g., cancer or an infectious disease) in a mammal, or to prevent a disease (e.g., cancer or an infectious disease) in a mammal are also provided. 
     In another aspect, the invention provides a population of bacteria comprising a plurality of  Listeria  bacteria, wherein each of the  Listeria  bacteria comprises a first polynucleotide comprising a polynucleotide encoding a holin protein. In some embodiments, the first polnucleotide further comprises a first promoter that is operably linked to the polynucleotide encoding the holin protein. In some embodiments, each of the  Listeria  bacteria further comprise (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide. The second polynucleotide may also optionally comprise a promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide. In some embodiments, the bacteria express the holin protein. In some embodiments, when the  Listeria  bacteria express the holin protein, expression of the holin protein does not substantially impair the net growth of the population. In some embodiments, when the  Listeria  bacteria express the holin protein, a substantial number of the  Listeria  bacteria are not lysed. In some embodiments, the  Listeria  bacteria comprises: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide. Pharmaceutical compositions, immunogenic compositions, and vaccines comprising the  Listeria  are further provided. In addition, methods of using the  Listeria  to induce an immune response to an antigen in a mammal, to treat a disease (e.g., cancer or an infectious disease) in a mammal, or to prevent a disease (e.g., cancer or an infectious disease) in a mammal are also provided. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the second polynucleotide encodes an RNA transcript comprising an expression cassette derived from an ssRNA positive-strand virus, wherein the expression cassette encodes the heterologous polypeptide. In some embodiments, the second polynucleotide comprises a replicon derived from a ss RNA positive-strand virus, which has been adapted to encode the heterologous polypeptide (including, but not limited to, a tumor antigen, or infectious disease antigen). In some embodiments, the virus is from a family selected from the group consisting of Togaviridae, Flaviviridae, and Picornaviridae. In some embodiments, the virus is a togavirsu, flavivirus, pestivirus, and picornavirus. In some embodiments, the RNA transcripts are capable of cap-independent translation. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, when the holin protein is expressed by the bacterium or bacteria, at least some of the second polynucleotide, heterologous polypeptide, RNA transcript of the second polynucleotide, and/or self-replicating RNA is released from the bacterium or bacteria. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the heterologous polypeptide does not comprise a signal peptide sequence. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  do not express a lysin protein. In some embodiments, the  Listeria  do not contain a polynucleotide comprising a polynucleotide (e.g., a recombinant polynucleotide) encoding a lysin protein. 
     In some alternative embodiments of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  comprise a polynucleotide comprising a polynucleotide (e.g., a recombinant polynucleotide) encoding a lysin protein. In some embodiments, the polynucleotide comprising the polynucleotide encoding the lysin protein is operably linked to a promoter. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the bacterium or bacteria express the holin protein when the bacterium is in the cytosol of an infected host cell. 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the first and/or second polynucleotide is in the genomic DNA of the  Listeria  bacterium. In some alternative embodiments, the first and/or second polynucleotide is on a plasmid. 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the first promoter is a prfA-dependent promoter (including, but not limited to, an actA promoter). 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the second promoter is a eukaryotic promoter. In some alternative embodiments, the promoter is a prokaryotic promoter. 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the holin protein is expressed in the bacterium when the bacterium or bacteria is in the cytosol of a host cell. 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the heterologous polypeptide comprises an antigen, such as a tumor antigen, or an antigenic fragment or variant thereof, or an infectious disease antigen, or an antigenic fragment or variant thereof. 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  are  Listeria monocytogenes.  In some embodiments, the  Listeria  is an attenuated form of  Listeria.    
     Methods of producing the  Listeria  described herein, as well as reagents useful in in the production of the  Listeria  such as parental  Listeria  strains and polynucleotides (e.g., expression cassettes) are also provided. 
     Further descriptions of the aspects and embodiments described above, as well as additional embodiments and aspects of the invention are provided below. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1A . Schematic drawing of virus-based, self-replicating, expression cassette, and holin mediated release of a nucleic acid encoding the expression cassette. 
         FIG. 1B . Schematic drawing of virus-based, self-replicating, expression cassette, and holin-mediated release of the expression cassette. 
         FIG. 1C . Schematic drawing of virus-based, self-replicating, expression cassette, and holin mediated release of the expression cassette. 
         FIG. 1D . Schematic drawing of holin mediated release of a macromolecule from a bacterium. 
         FIG. 2 . Schematic diagrams of plasmids containing expression cassettes encoding holin, lysin or holing and lysin. 
         FIG. 3A . Bacterial growth curves. 
         FIG. 3B . Bacterial growth curves. 
         FIG. 3C . Schematic diagrams of nucleic acids. 
         FIG. 3D . Bacterial growth curves. 
         FIG. 3E . Bacterial growth curves. 
         FIG. 3F . Bacterial growth curves. 
         FIG. 4 . Photographs showing fluorescent-stained bacteria and actin. 
         FIG. 5A . Schematic diagram of a plasmid pBHE573. 
         FIG. 5B . Holin mediated release of a DNA plasmid encoding luciferase out of a  Listeria  bacterium. 
         FIG. 6A . Schematic diagrams of plasmids pSH263 and pBHE530. 
         FIG. 6B . Holin mediated release of a DNA plasmid containing an alphavirus based, self replicating, expression vector (replicon) out of a  Listeria  bacterium. 
         FIG. 6C . Photographs of Lm-holin and Lm-holin-lysin infected mammalian cells. 
         FIG. 6D . Quantitation of Lm-holin and Lm-holin-lysin infected mammalian cells. 
         FIG. 6E . Lm ΔuvrAB-holin-lysin and KBMA Lm ΔuvrAB-holin-lysin infected mammalian cells. 
         FIG. 7 . Mammalian cells infected with Lm containing plasmids which include an IRES. 
         FIG. 8A . Schematic diagram of a virus based, self-replicating, expression cassette (replicon) that contains a 5′ terminal DI 25 structure, an IRES, and an open reading frame. 
         FIG. 8B . RNA containing an IRES electroporated into mammalian cells. 
         FIG. 9 . Infection of BHK cells with Lm strains expressing holin, lysin, or holin and lysin for delivering a cap-independent viral based replicon to the cytoplasm. 
         FIG. 10A . Infection of BHK cells with Lm strains expressing holin and lysin for delivering a cap-independent viral based replicon to the cytoplasm. 
         FIG. 10B . Quantitation of infection of BHK cells with Lm strains expressing holin and lysin for delivering a cap-independent viral based replicon to the cytoplasm 
         FIG. 11A . SIINFEKL-specific immune response. 
         FIG. 11B . LLO190-201-specific immune response. 
         FIG. 12A . Schematic diagram of plasmid pBHE558. 
         FIG. 12B . Holin mediated release of a polypeptide from Lm holin. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     I. General 
     The invention is based, in part, on the recognition that a  Listeria  bacterium engineered to contain a nucleic acid encoding a holin can mediate release of a nucleic acid from the bacterium. In some embodiments, the ability of  Listeria  to serve as a delivery vector can be improved by engineering the  Listeria  bacterium to contain a nucleic acid encoding a holin. In some embodiments of the invention, the holin permeabilizes the bacterial membrane, allowing release from the bacterium of antigens and nucleic acids encoding antigens. What is also encompassed in some embodiments is a  Listeria  bacterium containing a viral derived expression cassette, which may be released from the bacterium to the host cell&#39;s cytosol. With release, the expression cassette replicates and self amplifies, and expresses enhanced quantities of antigen. 
     In one aspect, the invention provides a  Listeria  bacterium (e.g.,  Listeria monocytogenes ) comprising a first polynucleotide comprising a polynucleotide encoding a holin protein. In some embodiments, the bacterium further comprises a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein. In some embodiments, the bacterium further comprises a second polynucleotide comprising a polynucleotide encoding a heterologous polypeptide (e.g., a heterologous polypeptide comprising an antigen). The second polynucleotide may further comprise a promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide. 
     In another aspect, the invention provides a population of bacteria comprising a plurality of  Listeria  bacteria, wherein each of the  Listeria  bacteria comprises a first polynucleotide comprising a polynucleotide encoding a holin protein. In some embodiments, the first polynucleotide further comprises a first promoter that is operably linked to the polynucleotide encoding the holin protein. In some embodiments, each of the  Listeria  bacteria further comprise (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide. The second polynucleotide may also optionally comprise a promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide. In some embodiments, when the  Listeria  bacteria express the holin protein, expression of the holin protein does not substantially impair the net growth of the population. In some embodiments, when the holin protein is expressed, the holin protein is expressed at a level that does not substantially impair the net growth of the population. In some embodiments, when the  Listeria  bacteria express the holin protein, a substantial number of the  Listeria  bacteria are not lysed. 
     In another aspect, the invention provides a  Listeria  bacterium, comprising: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide, wherein when the bacterium expresses the holin protein, expression of the holin protein does not substantially impair the growth of the bacterium. 
     In another aspect, the invention provides a  Listeria  bacterium, comprising: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide, wherein when the  Listeria  bacterium expresses the holin protein, the cell membrane of the bacterium is not lysed. 
     In still another aspect, the invention provides a  Listeria  bacterium, comprising: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein, and wherein the holin protein is derived from a non-listerial bacterium or from a bacteriophage that is not a listeriophage; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the bacterium or bacteria further comprise RNA transcripts generated from the second polynucleotide, wherein the RNA transcripts encode the heterologous polypeptide, and wherein, when the holin protein is expressed by the bacterium, at least some of the RNA transcripts are released from the bacterium, wherein the release is holin-dependent. In some embodiments, the RNA transcripts comprise an expression cassette derived from an ssRNA positive-strand virus, wherein the expression cassette encodes the heterologous polypeptide. 
     In a further aspect, the invention provides a population of bacteria comprising a plurality of  Listeria  bacteria, wherein each of the  Listeria  bacteria comprises: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide. 
     In a still further aspect, the invention provides a population of bacteria comprising a plurality of  Listeria  bacteria, wherein each of the  Listeria  bacteria comprises: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide, when the 
       Listeria  bacteria express the holin protein, expression of the holin protein does not substantially impair the net growth of the population. In some embodiments, when the  Listeria  bacteria express the holin protein, a substantial number of the  Listeria  bacteria are not lysed. 
     In another aspect, the invention provides a population of bacteria comprising a plurality of  Listeria  bacteria, wherein each of the  Listeria  bacteria comprises: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the heterologous polypeptide, and wherein, when the  Listeria  bacteria express the holin protein, a substantial number of the  Listeria  bacteria are not lysed. In some embodiments, when the  Listeria  bacteria express the holin protein, expression of the holin protein does not substantially impair the net growth of the population. 
     In a still further aspect, the invention provides a  Listeria  bacterium comprising: (a) a first polynucleotide comprising (i) a polynucleotide encoding a holin protein, and (ii) a first promoter, wherein the first promoter is operably linked to the polynucleotide encoding the holin protein; and (b) a second polynucleotide comprising (i) a polynucleotide encoding a self-replicating RNA, wherein the self-replicating RNA comprises a polynucleotide encoding a heterologous polypeptide, and (ii) a second promoter, wherein the second promoter is operably linked to the polynucleotide encoding the self-replicating RNA. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the polynucleotide(s) encoding the holin protein are recombinant polynucleotide(s). 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  bacteria express the holin protein. In some embodiments, expression of the holin protein occurs when the bacteria are in a host cell that they have infected (e.g., in the cytosol of an infected host cell). In some embodiments, the expression of the holin protein occurs only when the  Listeria  are in the cytosol of an infected host cell (e.g., a mammalian cell). 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, when the holin protein is expressed by the  Listeria,  the second polynucleotide, an RNA transcript generated from the second polynucleotide (e.g., an mRNA, self-replicating RNA, or expression cassette derived from an ssRNA positive-strand virus), and/or the heterologous polypeptide encoded by the second polynucleotide is released from the  Listeria  in a holin-dependent manner. For instance, in some embodiments, when the  Listeria  further comprise the heterologous polypeptide and the holin protein is expressed by the  Listeria,  at least some of the heterologous polypeptide is released from the  Listeria,  wherein the release is holin-dependent. In some embodiments, the released heterologous polypeptide does not comprise a signal peptide sequence. In some embodiments, when the holin protein is expressed by the  Listeria,  the second polynucleotide is released from the  Listeria,  wherein the release is holin-dependent. In some embodiments, when the  Listeria  further comprises a self-replicating RNA and the holin protein is expressed by the  Listeria,  at least some of the self-replicating RNA is released from the  Listeria,  wherein the release is holin-dependent. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  further comprise a third polynucleotide comprising a polynucleotide (e.g. a recombinant polynucleotide) encoding a lysin protein. In some embodiments, the third polynucleotide further comprises a promoter operably linked to the polynucleotide encoding the lysin protein. In some alternative embodiments, the  Listeria  do not express a Lysin protein and/or comprise a polynucleotide (e.g. a recombinant polynucleotide) encoding a Lysin protein. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the first, second and/or third polynucleotide (if present) resides in the genomic DNA of the  Listeria.  Alternatively, the first, second, and/or third polynucleotide (if present) resides on a plasmid. In some embodiments, the first, second, and/or third polynucleotides are parts of the same polynucleotide molecules. In some embodiments, the first, second, and/or third polynucleotides are contained on separate polynucleotide molecules. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide is a eukaryotic promoter. Alternatively, the promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide is a prokaryotic promoter. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the promoter that is operably linked to the polynucleotide encoding the heterologous polypeptide, the holin protein, and/or the lysin protein is a prfA-dependent promoter (e.g., an actA promoter). 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  is  Listeria monocytogenes.  In some embodiments, the  Listeria  is attenuated for cell-to-cell spread and/or entry into nonphagocytic cells. In some embodiments, the  Listeria  comprises an inactivating mutation in actA and/or inlB. In some embodiments, the  Listeria  is an actAinlB double deletion mutant. In some embodiments, the  Listeria  comprises an inactivating mutation in at least one nucleic acid repair gene, such as uvrA, uvrB, uvrC, or a recombinational repair gene. For instance, the  Listeria  may be a uvrAB deletion mutant. In some embodiments, the bacterium further comprises a nucleic acid cross-linking agent (e.g., a psoralen). 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the heterologous polypeptide comprises an antigen. The antigen may be a tumor antigen, or an antigenic fragment or variant thereof. Alternatively, the antigen may be an antigen from an infectious agent, or an antigenic fragment or variant of such an antigen. 
     In some embodiments, of each of the aforementioned aspects, as well as other aspects described herein, the RNA transcripts of the polynucleotide encoding the heterologous protein and/or the self-replicating RNAs comprise an expression cassette derived from an ssRNA positive-strand virus, wherein the expression cassette encodes the heterologous polypeptide. In some embodiments, the expression cassette that is derived from an ssRNA positive-strand virus comprises a replicon derived from the virus. In some embodiments, the replicon derived from the ssRNA positive-strand virus comprises sufficient genetic elements from the genome of that virus to allow for self-amplification or self-replication of the expression cassette encoding the heterologous polypeptide in a eukaryotic cell, such as a mammalian cell. In some embodiments, the virus is a virus from a family selected from the group consisting of Togaviridae, Flaviviridae, and Picornaviridae. In some embodiments, the virus is a virus selected from the group consisting of togavirus (e.g., an alphavirus), flavivirus (e.g., Kunjin virus or yellow fever virus), pestivirus (e.g., Bovine Viral Diarrhea Virus), and picornavirus (e.g., Encephalomyocarditis (EMCV) virus, poliovirus, or coxsackie virus). In some embodiments, the self-replicating RNA comprises an alphavirus replicon that expresses the heterologous polypeptide. In some embodiments, the alphavirus replicon is derived from Sindbis virus, Venezuelan Equine Encephalitis (VEE) virus, or Semliki Forest virus (SFV). In some embodiments, the self-replicating RNA comprises a picornavirus replicon that expresses the heterologous polypeptide. In some embodiments, the picornavirus replicon is derived from poliovirus. In some embodiments, the self-replicating RNA comprises a flavivirus replicon that expresses the heterologous polypeptide. In some embodiments, the self-replicating RNA is derived from Bovine Viral Diarrheal Virus (BVDV). In some embodiments, the RNA transcripts and/or self-replicating RNAs are capable of cap-independent translation (e.g., contain an IRES). 
     In some embodiments, the  Listeria  express holin and lysin and the polynucleotide encoding the heterologous polypeptide comprises a replicon derived from a poliovirus. In some embodiments, the  Listeria  express holin, but not lysin, and the polynucleotide encoding the heterologous polypeptide comprises a replicon derived from a sindbis virus. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the  Listeria  bacterium or bacteria further expresses a viral protein that suppresses the type I interferon (IFN) response in infected host cells upon phagosomal escape into the host cell cytoplasm. Non-limiting examples of such viral proteins are provided, e.g., in Table 11 and Example 6, below. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, greater than 10%, greater than 25%, greater than 50%, greater than 75%, or greater than 99% of the expressed antigen is released from the from the  Listeria  into the cytoplasm of a eukaryotic cell upon escape of the  Listeria  from the phagolysosome. 
     In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, release of the antigen from the  Listeria  into the cytoplasm of a eukaryotic cell upon escape of the  Listeria  from the phagolysosome is greater than 10% dependent on the expressed holin, greater than 25%, greater than 50%, greater than 75%, or greater than 99% dependent on the expressed holin. 
     Pharmaceutical compositions, immunogenic compositions, and/or vaccines comprising the  Listeria  of the aforementioned aspects and embodiments are further provided. In addition, methods of using the  Listeria  to induce an immune response to an antigen in a mammal or to treat or prevent a disease (e.g., cancer or a non-listerial infectious disease) in a mammal are also provided. In some embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. In some embodiments, the  Listeria  further comprise an adjuvant. 
     The invention further provides bacterial populations comprising the  Listeria  described herein. In some embodiments, the  Listeria  described herein make up at least about about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 98% of the  Listeria  bacteria in a given population. In some embodiments, the  Listeria  described herein make up at least about about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 98% of the total bacteria in a given population. 
     In some embodiments, expression of the holin protein does not substantially impair the growth of a bacterium or the net growth of a population of bacteria. The growth of a bacterium or the net growth of a population of bacteria is said to not be substantially impaired if the growth rate is not decreased by more than about 2-fold relative to an appropriate reference or control (e.g., a parental strain). In some embodiments, the rate of growth is not decreased by more than about 10%, more than about 20%, more than about 30%, or more than about 40% (relative to an appropriate control). For instance, expression of a holin protein does not substantially impair the growth of a bacterium that expresses the holin protein (or the net growth of a population of bacteria expressing the holin protein) if the growth of the bacterium (or the net growth of the population of bacteria) is not decreased by more than about 2-fold relative to a control such as a bacterium (or bacterial population) that is not expressing the holin protein, but is otherwise generally equivalent. Generally, the growth of the bacteria are compared under identical environmental conditions, although in some instances growth following induction of expression of holin may be compared to growth prior to induction of expression of holin. In some embodiments, the control is a bacterium (or bacterial population) that does not comprise the polynucleotide encoding the holin protein. In some embodiments, the growth or net growth that is measured and compared is intracellular growth, i.e., growth in cells, such as mammalian cells (e.g., J774 cells). In some embodiments, the growth or net growth in the cytoplasm of mammalian cells is measured. Intracellular growth of a  Listeria  bacterium or population can be measured by light microscopy, fluorescent microscopy, colony forming unit (CFU) assays, or the quantity of listerial antigens or  Listeria -specific sequences. In some embodiments, the growth or net growth is growth in broth culture or on agar. Growth in broth culture can, e.g., be measured by OD 600 . 
     The identification of  Listeria  as lysed can, e.g., be made microscopically. The number of  Listeria  in a population that are lysed can likewise be determined microscopically. Alternatively, the level of lysis occurring within a bacterial population can instead be measured indirectly by measuring the net growth rate of the population. A substantial number of  Listeria  in a population are said to not be lysed when at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells within an observed population are not lysed. 
     In some embodiments, the  Listeria  may comprise multiple copies of the polynucleotides encoding the holin protein. For instance, the  Listeria  may comprise two polynucleotides encoding the holin protein. 
     The term “polynucleotide” is used interchangeably herein with “nucleic acid.” 
     II. Holin and Lysin Proteins and Encoding Polynucleotides 
     In some embodiments, the  Listeria  described herein comprise a polynucleotide encoding a holin protein and/or express a holin protein. In some embodiments, the polynucleotide encoding the holin protein is recombinant. In some embodiments, the  Listeria  comprise a polynucleotide encoding a lysin protein and/or express a lysin protein. In some embodiments, the  Listeria  do not comprise a polynucleotide encoding a lysin protein and/or do not express a lysin protein. In certain embodiments, the polynucleotide encoding the lysin protein is recombinant. In some embodiments, the polynucleotides encoding the holin and/or lysin are operably linked with a promoter specifically activated when the  Listeria  is inside a host mammalian cell (e.g., actA promoter). 
     The terms “holin proteins” and “holins” (used interchangeably herein) encompass membrane proteins that are capable of permeabilizing the cytoplasmic membrane of bacteria. Holins may also facilitate the activity of lysins against the peptidoglycan. The terms “lysin proteins” and “lysins” (used interchangeably herein) encompass enzymes that degrade the cell wall peptidoglycan. Lysins may have activity against the glycosidic, amide, or peptide bonds of the cell wall. Holin proteins and lysin proteins need not necessarily be full-length proteins. Thus, the holin proteins or lysin proteins encompass fragments or variants of naturally occurring holin proteins or lysin proteins, respectively, so long as the fragments or variants are functionally active. In some embodiments, holins and/or lysins are phage-encoded. In some embodiments, the sequences encoding the holins and/or lysins may have been identified in bacterial genomes. 
     In some embodiments, expression of a holin by  Listeria  results in permeabilization of the  Listerial  membrane, where the permeabilized membrane can allow the release of non-secretory proteins, secretory proteins (that have not yet been secreted), large and small polypeptides, nucleic acids encoding heterologous antigens, virus-derived expression cassettes, and the like. 
     By way of non-limiting examples, the  Listeria  of the invention can contain a nucleic acid encoding one holin, for example, a recombinant holin, a holin operably linked with a promoter specifically activated inside a host mammalian cell, or a holin operably linked with a prfA-dependent promoter. Moreover, the  Listeria  of the invention can contain a nucleic acid encoding a single transcription unit that encodes two copies of the same holin. Also, the  Listeria  can contain a nucleic acid encoding a single transcription unit that encodes copies of two different holin, for example, from two different types of listeriophages. To give another example, the  Listeria  can contain two nucleic acids that encode two different transcriptional units, where each transcriptional unit encodes a holin, and where the two holins can be the same or different. The invention contemplates  Listeria  encoding one, two, three, or more holins. In one aspect, a nucleic acid encoding a holin is integrated into a gene encoding a virulence factor, where the nucleic acid is operably linked to the promoter(s) of the virulence factor gene. In another aspect, a nucleic acid encoding a lysin is integrated into a gene encoding a virulence factor, where the nucleic acid is operably linked to the promoter(s) of the virulence factor gene. 
     A number of holins and lysins have been expressed in bacteria, as demonstrated by studies of substrate specificity of the holin (the substrate being the lipid membrane of a specific bacterium), and substrate specificity of the lysin (the substrate being peptidoglycan of a specific bacterium). These studies have demonstrated that membrane permeabilization can be accomplished by a variety of holins, and is not limited to a holin expressed by a bacteriophage that happens to specifically infect that bacterium. Also, the studies have demonstrated that bacterial cell lysis can be accomplished by the combination of a holin from a first type of bacteriophage and a lysin from an unrelated, second type of bacteriophage. Nucleic acids encoding the holin and lysin can have an origin different from the bacterium used for the lysis study. For example, bacterial lysis can occur where the holin originates from a first type of phage, for example,  Lactobacillus gasseri  phage phi-adh, and the lysin originates from a second type of phage, for example,  Bacillus subtilis  phage phi-29 (see, e.g., Henrich, et al. (1995) J. Bacteriol. 177:723-732). Bacterial lysis can occur where the holin and lysin originate from a phages that do not or cannot infect the bacterium (see, e.g., Henrich, et al. (1995) J. Bacteriol. 177:723-732; Grundling, et al. (2000) J. Bacteriol. 182:6075-6081; Steiner, et al. (1993) J. Bacteriol. 175:1038-1042; Berkmen, et al. (1997) J. Bacteriol. 179:6522-6524; Loessner, et al. (1999) J. Bacteriol. 181:4452-4460). 
     Control of the rate of lysis, for example, delayed lysis, can be accomplished by a number of methods. The rate of lysis can be altered by a promoter specifically activated inside a mammalian host cell that is operably linked with a nucleic acid encoding a holin. Alternatively, altered rates of bacterial cell lysis can be accomplished by expressing a nucleic acid encoding a holin that harbors a specific mutation, by expressing selected levels of a holin inhibitor (see, e.g., Grundling, et al. (2000) J. Bacteriol. 182:6082-6090), or by using a nucleic acid encoding a slower acting holin, such as phi 29 protein 14 (holin), or a faster acting holin, such as phi adh holin (see, e.g., Henrich, et al. (1995) J. Bacteriol. 177:723-723). 
     In some embodiments, a holin protein encoded by a polynucleotide (e.g., a recombinant polynucleotide) in the  Listeria  comprises, or is derived from, one of the following holins: 
     
       
         
           
               
            
               
                  1- Listeria innocua  Clip11262: Lin1702 
               
               
                 &gt;gi|16800770|ref|NP_471038.11 hypothetical protein 
               
               
                 lin1702 [ Listeria innocua  Clip11262] 
               
            
           
           
               
            
               
                 (SEQ ID NO: 1) 
               
            
           
           
               
            
               
                 MKINWKVRMKSKVFWVSVIPLVLVLAQQLLGWFGVTIPADTVNKQALDFV 
               
               
                   
               
               
                 NSVFLLLGVLGVVNDPTTEGTADSELVLNRNRKDEE; 
               
               
                   
               
               
                  2- Lactococcus lactis  subsp.  cremoris  SK11: 
               
               
                 Llacc01000415 
               
               
                 &gt;gi|62464385|ref|ZP_00383678.1|COG5546: Small 
               
               
                 integral membrane protein [ Lactococcus lactis   
               
               
                 subsp.  cremoris  SK11] 
               
            
           
           
               
            
               
                 (SEQ ID NO: 2) 
               
            
           
           
               
            
               
                 MNQINWKLRLKSKAFWLALLPALFLLIQAIGAPFGYKWDFVILNQQLAAV 
               
               
                   
               
               
                 VNAAFALLAIVGVVSDPTTSGLGDSDRVLNKDKSEENK; 
               
               
                   
               
               
                  3- Enterococcus faecalis  V583: EF1993 
               
               
                 &gt;gi|29376514|ref|NP_815668.1|holin [ Enterococcus   
               
               
                   faecalis  V583] 
               
            
           
           
               
            
               
                 (SEQ ID NO: 3) 
               
            
           
           
               
            
               
                 MINWKSRIKNKQFWLSIIPAVLLLIQVVAVPFGYKFQIEMINKQLLDVVN 
               
               
                   
               
               
                 ALFVVLTILGIVTDPTTPGLSDRKGDK; 
               
               
                   
               
               
                  4- Staphylococcus aureus  subsp.  aureus  Mu50: 
               
               
                 SAV1946 
               
               
                 &gt;gi|15924936|ref|NP_372470.1|holin homolog 
               
               
                 [ Staphylococcus aureus  subsp.  aureus  Mu50]; phage 
               
               
                 phi LC3 
               
            
           
           
               
            
               
                 (SEQ ID NO: 4) 
               
            
           
           
               
            
               
                 MINWKIRMKQKSFWVAILSAIFLFAQNIAKAIGYDIQVYTEQLTDGLNAI 
               
               
                   
               
               
                 LGFLVLTGVIQDPTTKGIGDSHQALEYEEPRRKY; 
               
               
                   
               
               
                  5- Bacillus licheniformis  ATCC 14580: BL01378 
               
               
                 &gt;gi|52082617|ref|YP_081408.11 hypothetical protein 
               
               
                 BL01378 [ Bacillus licheniformis  ATCC 14580] 
               
            
           
           
               
            
               
                 (SEQ ID NO: 5) 
               
            
           
           
               
            
               
                 METVLIFASVLSPIILALVELVKKTVKMPKNLIPLVSLLIGLLIGAAAYP 
               
               
                   
               
               
                 FTDLELVLRLWSGGLAGLTATGLFEIGKNRNARKKKNP; 
               
               
                   
               
               
                  6- Bacillus anthracis  Sterne: BAS3785 
               
               
                 &gt;BAS3785 
               
            
           
           
               
            
               
                 (SEQ ID NO: 6) 
               
            
           
           
               
            
               
                 MDRIDVLLKAFIATFGGFCGYFLGGWDATLKILVTMAVIDYLTGMIAAGY 
               
               
                   
               
               
                 NGELKSKVGFKGIAKKVVLFLLVGAAAQLDSALGSNSAIREATIFFFMGN 
               
               
                   
               
               
                 ELLSLLENAGRMGIPLPQALTNAVEILGGKQKQEEKKGDVE; 
               
               
                   
               
               
                  7- Clostridium perfringens  13: CPE0383 
               
               
                 &gt;CPE0383 
               
            
           
           
               
            
               
                 (SEQ ID NO: 7) 
               
            
           
           
               
            
               
                 MEGIIICIKLGVVFLGTLFTWIFGAWDMPIVTLLVFIFLDYLTGVIKGCK 
               
               
                   
               
               
                 SKELCSNIGLRGITKKGLILVVLLVAVMLDRLLDNGAWMFRTLIAYFYIM 
               
               
                   
               
               
                 NEGISILENCAALGVPIPEFLRQALKQLNNKNNIK; 
               
               
                   
               
               
                  8- Corynebacterium diphtheriae  NCTC13129: DIP2153 
               
            
           
           
               
            
               
                 (SEQ ID NO: 8) 
               
            
           
           
               
            
               
                 MPVKPASPRSHPGCPELTHERYCDAQAKAEDARYRKYQRDPKINRRYGSR 
               
               
                   
               
               
                 WRKIRAAYVAAHPLCEDCLEAGRYTPVQEVHHVLPIEHGGTHNFDNLQSL 
               
               
                   
               
               
                 CKPCHSRQSALDDDRWRQQPRVYTY; 
               
               
                   
               
               
                  9- Lactobacillus johnsonii  NCC 533: NT01LJ1229 
               
               
                 &gt;LJ_1419 
               
            
           
           
               
            
               
                 (SEQ ID NO: 9) 
               
            
           
           
               
            
               
                 MSVNQLLDLSIVVVSVAAVVVASVYAKHKIAIDKKAAQGDLLAKAEKIVA 
               
               
                   
               
               
                 QSVSPLVYQAEKRGGDGEDKLTFVVQGLFLLLDMAHLPHPTMSFVKGMVE 
               
               
                   
               
               
                 KSVTAMKQAQSIADTVDKPKPTVVGELREVKK; 
               
               
                 and/or 
               
               
                   
               
               
                 10- Streptococcus agalactiae  2603V/R: SAG1838 
               
               
                 &gt;SAG1838 
               
            
           
           
               
            
               
                 (SEQ ID NO: 10) 
               
            
           
           
               
            
               
                 MTQITDIIISSAMGILTILAGIAVQAVKVYLIKKGGEKAVLITEILAKNA 
               
               
                   
               
               
                 VNAVEQVATETGFKGADKLTSAKAQILAELQKYNIHMSDDDLTLFVESAV 
               
               
                   
               
               
                 KQMHDAWKE. 
               
            
           
         
       
     
     Without implying any limitation, the invention provides a  Listeria  containing a first nucleic acid encoding holin, a second nucleic acid encoding a lysin, or both the first and second nucleic acids, where the nucleic acids are from one or more listeriophages. The listeriophages can be, for example, A118, PSA, A511, B054, B024, B055, C707, B053, B051, D441, B604, A020, B025, B545, B653, A500, A006, B101, B056, B012, B110, B035, A502, 9425, 1313, 197, 12682, 6223, 5775, 10, 43, 43, 21, 19, 387, 1967, 2685, 4477, 575, 1652, 12029, 52, 340, 312, 108, 107, 47, 2671, 1444, 2425, 3551, 3552, 1317, 2389, 3274, 9495, 1313, 197, A511, 6223, 12682, 5775, and the like. Also provided are nucleic acids encoding a holin, lysin, or both holin and lysin, from listeriophage A511, A118, A502, A006, B653, B054 (4286), B051 (4295), B025, D441, B545, B053 (4277), B056 (5337), B101, B110, C707, B024, B012, B035, A020, A500, 4211, 2671, and 2389 (see, e.g., Zink and Loessner (1992) Appl. Environ. Microbiol. 58:296-302; Mee-Marquet, et al. (1997) Appl. Environ. Microbiol. 63:3374-3377; Loessner (1991) Appl. Environ. Microbiol. 57:882-884; Zink, et al. (1995) Microbiology 141:2577-2584; Loessner, et al. (1994) J. Gen. Virol. 75:701-710; Loessner, et al. (1994) Intervirol. 37:31-35; Ackermann, et al. (1981) Ann. Virol. (Inst. Pasteur) 132E:371-382; Chiron, et al. (1977) C.R. Soc. Biol. (Paris) 171:488-491; Ortel and Ackermann (1985) Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. Reihe A 260:423-427; Rocourt, et al. (1983) Ann. Virol. (Inst. Pasteur) 134E:245-250; Rocourt, et al. (1985) Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. Reihe A 259:341-350). Moreover, what is provided is EJ-1 phage holin or lysin (Haro, et al. (2003) J. Biol. Chem. 278:3929-3936). 10091] The holin is able to mediate transfer of a reagent or substance from inside the  Listeria  bacterium, through the lipid membrane and cell wall, to the exterior of the  Listeria  bacterium. The transfer-mediating function of the holin can be measured, for example, by release of a plasmid encoding luciferase or another marker, a replicon, an antigen, or a fluorescent marker. Methods for assessing permeability and pore diameters are available (see, e.g., Dijkstra and Keck (1996) J. Bacteriol. 178:5555-5562; Pink, et al. (2000) J. Bacteriol. 182:5925-5930; Sara and Sleytr (1987) J. Bacteriol. 169:4092-4098; Demchick and Koch (1996) J. Bacteriol. 178:768-773). 
     The invention encompasses non-listeriophage holins, including  Serratia marcescens  NucE (Berkmen, et al. (1997) 179:6522-6524);  Staphylococcus aureus  bacteriophage 187 holin (Loessner, et al. (1999) J. Bacteriol. 181:4452-4460; phage lambda holins and  Lactobacillus gasseri  phi-adh holin (Henrich, et al. (1995) J. Bacteriol. 177:723-732; phage phi-29 holin (Steiner, et al. (1993) J. Bacteriol. 175:1038-1042);  Bacillus  phage PZA holin (Loessner, et al. (1997) J. Bacteriol. 179:2845-2851); phage T4 gpt holin (Dressman and Drake (1999) J. Bacteriol. 181:4391-4396); phage PRD1 holin (Ziedaite, et al. (2005) J. Bacteriol. 187:5397-5405);  Borrelia burgdorferi  prophage BlyA (Damman, et al. (2000) J. Bacteriol. 182:6791-6797);  Bacillus subtilis  ywcE holin (Real, et al. (2005) J. Bacteriol. 187:6443-6453);  Staphylococcus aureus  lrgA holin and cidA holin (Brunskill and Bayles (1996) J. Bacteriol. 178:5810-5812; Rice, et al. (2004) J. Bacteriol. 186:3029-3037);  Streptococcus pneumoniae  cph1 holin, pneumococcal phage EJ-1 holin, phi-LC3 holin and Tuc2009 holin of  Lactococcus lactis  phage (Martin, et al. (1998) J. Bacteriol. 180:210-217); bacteriophage P2 gene Y holin (Ziermann, et al. (1994) J. Bacteriol. 176:4974-4984); and bacteriophage PRD1 holin P35 (Rydman and Bamford (2003) J. Bacteriol. 185:3795-3803). 
     Nucleic acids in bacterial genomes encoding proteins identified as holins are available (Table 1). Some of these holins can be characterized as bacterial holins, that is, holins that are not holins of cryptic phages. A cryptic phage is a phage genome integrated in the bacterial genome. These holin genes include the CidA gene of  S. aureus  (see, e.g., Rice, et al. (2003) J. Bacteriol. 185:2635-2643; Rice and Bayles (2003) Mol. Microbiol. 50:729-738; Bayles (2000) Trends Microbiol. 8:274-278; GenBank Acc. No. AY581892). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Bacterial genomic nucleic acids encoding holins. 
               
            
           
           
               
               
            
               
                 Bacterium 
                 GenBank Acc. No. 
               
               
                   
               
               
                 
                   Bacillus subtilis 
                 
                 Z99117, nt 51006-51428. 
               
               
                 
                   Bacillus subtilis 
                 
                 NC_000964, nt 2263876-2264088; 
               
               
                   
                 3932232-3932618. 
               
               
                   Bacillus anthracis  strain Sterne 
                 NC_005945, five holins, e.g., 
               
               
                   
                 3432919-3433284. 
               
               
                 
                   Pseudomonas entomophila 
                 
                 NC_008027, compl. nt 
               
               
                   
                 4463886-4464239. 
               
               
                 
                   Escherichia coli 
                 
                 BA000007, ten holins, e.g., 
               
               
                   
                 nt 901806-902021. 
               
               
                   Listeria monocytogenes  strain 
                 NC_002973 nt 142006-142428. 
               
               
                 4b F2365 
               
               
                 
                   Listeria innocua 
                 
                 AL596169, nt compl. 165378-165638. 
               
               
                 
                   Staphylococcus epidermidis 
                 
                 CP000029, nt 2047024-2047482. 
               
               
                 
                   Erwinia carotovora 
                 
                 NC_004547, compl. nt 
               
               
                   
                 2950159-2950467. 
               
               
                 
                   Corynebacterium diphtheriae 
                 
                 NC_002935, six holins, e.g., 
               
               
                   
                 nt 3637616-3637981. 
               
               
                 
                   Corynebacterium diphtheriae 
                 
                 BX248360, compl. nt 129273-129650. 
               
               
                 
                   Staphylococcus aureus 
                 
                 AJ938182, four holins, e.g., compl. 
               
               
                   
                 1846356-1846793. 
               
               
                 
                   Salmonella typhimurium 
                 
                 AE008823 nt 16310-16529. 
               
               
                 
                   Rhodopseudomonas palustris 
                 
                 BX572594, nt 258068-258451. 
               
               
                   
               
            
           
         
       
     
     Lysin-encoding nucleic acids are available. Lysins from listeriophage A118 (ply118 lysin), listeriophage A500 (ply500 lysin), and listeriophage 2438 (Cp12438 lysin), from the  L. monocytogenes  EGDe genome, and lysins designated as L-alanyl-D-glutamate peptidases, are available (Loessner, et al. (2002) Mol. Microbiol. 44:335-349; Glaser, et al. Science 294:849-8521; Zink, et al. (1995) Microbiology 141:2577-2584; Loessner, et al. (1995) Mol. Microbiol. 16:1231-1241). The contemplated lysins encompass catalytically active lysins that are mutated, e.g., occurring as fusion proteins, truncated proteins, amino acid substituted, and amino acid deleted proteins, and the like. 
     What is also contemplated is a  Listeria  containing a nucleic acid encoding a lysin, where the lysin is, or is derived from, e.g., listeriophage A500 (GenBank Acc. No. X85009);  Listeria innocua  clip11262 (GenBank Acc. Nos. AL596169; AL596172, AL596163);  Listeria innocua  (GenBank Acc. No. X89234);  Bacillus licheniformis  ATCC 14580 (GenBank Acc. Nos. CP000002; AE017333); listeriophage PSA (GenBank Acc. No. AJ312240); and related sequences. 
     Holin can be provided in combination with an endogenous lysin, for example, a lysin encoded by an integrated bacteriophage. Holin can be provided with a recombinant lysin, for example, as supplied by a recombinant nucleic acid in the bacterium or as supplied exogenously. Moreover, the holin can be provided in the absense of any lysin. 
     Table 2 discloses a number of lysins available for the invention. What is contemplated is a  Listeria  containing a nucleic acid encoding a lysin, or a lysin deleted in its secretory or membrane-associating sequence. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Nucleic acids encoding lysins. 
               
            
           
           
               
               
            
               
                 Lysin 
                 Citation 
               
               
                   
               
               
                   Listeria  murA 
                 Carroll, et al. (2003) J. Bacteriol. 185: 6801-6808; GenBank Acc. No. 
               
               
                 peptidoglycan 
                 NC_002973 (complement of nt 2558636-2559928); GenBank Acc. No. 
               
               
                 hydrolase 
                 AM039955; GenBank Acc. No. NC003212 (complement of 
               
               
                   
                 ny 2688314-2689606); GenBank Acc. No. NC_003210 (complement of 
               
               
                   
                 nt 2603863-2605155). The signal peptide of murA is encoded by nt 601-757, 
               
               
                   
                 while mature murA protein is encoded by nt 758-2361 (GenBank Acc. No. 
               
               
                   
                 AM039955). 
               
               
                 
                   Pediococcus 
                 
                 Musacchio, et al. (2003) J. Appl. Microbiol. 94: 561-570); ply118 
               
               
                 peptidoglycan 
                 L-alanoyl-D-glutamate peptidase (see, e.g., Gaeng, et al. (2000) Appl. Environ. 
               
               
                 hydrolase 
                 Microbiology 66: 2951-2958); ply511 N-acetylmuramoyl-L-alanine amidase 
               
               
                   
                 (see, e.g., Gaeng, et al., supra). 
               
               
                 p60 (also 
                 Lenz, et al. (2003) Proc. Natl. Acad. Sci. USA 100: 12432-12437; GenBank Acc. 
               
               
                 known as 
                 Nos. DQ060360; NC_003210 (complement of nt 618932-620380); AF532220; 
               
               
                 CwhA; iap 
                 AF532218; M95579; GenBank Acc. No. AM040043; AE017262. Monk, et al. 
               
               
                 gene). 
                 (2004) Appl. Environ. Microbiology 70: 6686-6694; Bubert, et al. (1992) J. Bacteriol. 
               
               
                   
                 174: 8166-8171. 
               
               
                 FlaA (murein 
                 Popowska and Markiewicz (2004) Pol. J. Microbiol. 53: 237-241; Popowska 
               
               
                 hydrolase). 
                 (2004) Pol. J. Microbiol. 53: 29-34; GenBank Acc. No. AL591976 (nt 
               
               
                   
                 84881-85836 of segment 4/12); GenBank X65624; GenBank Acc. No. 
               
               
                   
                 NC_003212 (nt 724183-725046). 
               
               
                 Major 
                 Ouzari, et al. (2002) J. Appl. Microbiol. 92: 812-820. 
               
               
                 autolysin of 
               
               
                 
                   Lactococcus 
                 
               
               
                   lactis  (active 
               
               
                 against 
               
               
                   Listeria ). 
               
               
                 Ami (amidase; 
                 GenBank Acc. No. AL591983 (complement of nt 240167-242920 of genome 
               
               
                 autolysin). 
                 segment 11/12); Milohanic, et al. (2001) Mol. Microbiol. 39: 1212-1224. 
               
               
                 LysA. 
                 GenBank Acc. No. AF042193; McLaughlan and Foster (1998) Microbiol. 
               
               
                   
                 144: 1359-1367; McLaughlan and Foster (1997) FEMS Microbiol. Lett. 
               
               
                   
                 152: 149-154. 
               
               
                 Listerial 
                 Reinscheid, et al. (2001) J. Bacteriol. 183: 1175-1183; GenBank Acc. No. 
               
               
                 M45 peptidase 
                 NC_003210 (complement nt 2581900-2583105), and homologous listerial 
               
               
                 and 
                 proteins, lmo2505 gene (GenBank Acc. No. AL591983). 
               
               
                 homologous 
               
               
                 listerial 
               
               
                 proteins, 
               
               
                 lmo2505 gene. 
               
               
                 lin2647 gene 
                 GenBank Acc. No. AL596173, nt 54941-56254 of segment 11/12. 
               
               
                 M48 peptidase 
                 GenBank Acc. No. AE017262. 
               
               
                   
               
               
                 What is available for the invention is a nucleic acid encoding a homologous holin, or a homologous lysin, where the percent sequence identity to the parent polypeptide is normally at least 90%, more normally at least 80%, most normally at least 70%, typically at least 60%, more typically at least 50%, most typically at least 40%, conventionally at least 30%, and more conventionally at least 20% amino acid sequence identity. 
               
            
           
         
       
     
     What is available are nucleic acids encoding lysin enzymes active against listerial peptidoglycan (murein), for example, peptidoglycan hydrolase; murein hydrolase; endolysin; transglycosylase; endopeptidase; autolysin; lysozyme; N-acetylmuramidase; N-acetylmuramyl-L-alanine amidase (amidase); endo-β-N-acetylglucosaminidase (glucosaminidase); where the enzyme has the property of partially and/or substantially catalyzing the degradation of listerial peptidoglycan. The invention is not limited by the mechanism of murein degradation or modification, and can include mechanisms such as hydrolysis or transglycosylation (see, e.g., Carroll, et al. (2003) J. Bacteriol. 185:6801-6808; Heidrich, et al. (2002) J. Bacteriol. 184:6093-6099). Also encompassed are nucleic acids encoding lysins deleted in any secretory sequence or sorting signal that mediates cell wall attachment (see, e.g., Sabet, et al. (2005) Infection Immunity 73:6912-6922; Catt and Gregory (2005) J. Bacteriol. 187:7863-7865). 
     Regarding listerial constructs, in certain embodiments, nucleic acids encoding holin and/or lysin can be integrated at any position in a gene encoding a virulence factor, where integration results in an attenuating mutation. The  Listeria  can be engineered to contain a plurality of nucleic acids encoding a holin, where the plurality of nucleic acids can encode an identical holin, e.g., solely from listeriophage PSA, or different holins, e.g., from listeriophage and also from lambda phage. The plurality of nucleic acids can be integrated at the same locus in the listerial genome, integrated solely in the actA gene, or at different loci in the listerial genome, e.g., integrated in the actA gene and in the inlB gene. The two nucleic acids may be bicistronic. 
     The nucleic acid can be operably linked with a promoter that is specifically activated in a mammalian host cell, such as a prfA-activated promoter. Without implying any limitation, the promoter can be, or can be derived from, actA promoter, inlB promoter, plcA promoter, hly promoter (listeriolysin O; LLO), plcB promoter, prfA promoter, mpl promoter, and so on. 
     Efficacy of the holin and lysin embodiments of the invention, in mediating the processing and presentation of an antigen, can be assessed by a number of methods. These methods include, e.g., microscopy to monitor or detect antigen in a host cell&#39;s cytosol; methods of immunology sensitive to the processing or presentation of an antigen by an APC containing the  Listeria  (see, e.g., Porgador and Germain (1997) Immunity 6:715-726; Shastri and Gonzalez (1993) J. Immunol. 150:2724-2736); methods for measuring activation or proliferation of antigen-specific CD8 +  T cells or CD4 +  T cells; methods for measuring tumor size, infectious agent titer, and survival. Efficacy of the holin and lysin embodiments of the invention can also be assessed by measuring expression of the holin or lysin by the  Listeria  bacterium, residence of the holin within the membrane, e.g., by antibodies specific for holin, holin or lysin-mediated entry of a marker molecule from a medium into the  Listeria  bacterium, production of murein degradation products, and the like. 
     A number of  Listeria -compatible promoters are available for operable linkage with a nucleic acid encoding holin, lysin, or virus-derived expression cassette. The promoter can be one that is specifically activated inside a host mammalian cell, or one that is constitutive. PrfA-dependent promoters are specifically activated in a host cell. Available prfA-dependent promoters include the prfA promoter itself, as well as actA promoter, inlB promoter, orfX promoter, orfZ promoter, uhpT promoter, and the like (Gray, et al. (2006) Infection Immunity 74:2505-2512; Chatterjee, et al. (2006) Infection Immunity 74:1323-1338). What is available are combinations of the same prfA-dependent promoters, of different prfA-dependent promoters, and of prfA-dependent and prfA-independent promoters, that is, acting in tandem and operably linked with the same ORF. 
     The promoter can be from a non-listerial organism (see, e.g., U.S. Pub. No. US 2005/0249748, incorporated by reference herein in its entirety). It can be a hybrid of two different promoters, it can be partially synthetic, and it can be totally synthetic, that is, having little sequence identity to a naturally occurring promoter. 
     What is also available are the regulatory regions, including promoters, for any of the 301 listerial genes documented to be upregulated during intracellular growth, or any of the 115 genes upregulated for growth in the cytosolic compartment (Chatterjee, et al., supra). 
     III. Heterologous Polypeptides and Polynucleotides Encoding the Heterologous Polypeptides 
     “Heterologous polypeptides” that are encoded by polynucleotides within the  Listeria  and/or expressed by the  Listeria  are heterologous with respect to the  Listeria.  In certain embodiments, the heterologous polypeptides are non-listerial. In certain embodiments, the heterologous polypeptides are not found in  Listeria  in nature in either the genomic DNA or in any bacteriophage that has infected the  Listeria.  In some embodiments, the polynucleotides encoding the heterologous polypeptide(s) are recombinant. 
     In some embodiments, where the polynucleotide encoding the heterologous polypeptide is to be expressed within the  Listeria,  operably linked promoters capable of directing expression in  Listeria  are preferred. In some embodiments, the promoters are prokaryotic (e.g., listerial promoters such as the hly or actA promoters). In some embodiments, the polynucleotides encoding the heterologous antigen are codon-optimized for expression in  Listeria  (see, e.g., U.S. Patent Publication No. 2005/0249748, incorporated by reference herein in its entirety). 
     In some embodiments, where the polynucleotide encoding the heterologous polypeptide is to be expressed in the cytosol of an infected eukaryotic cell, such as a mammalian cell, operably linked promoters capable of directing expression in the cell are preferred. In some embodiments, the promoters are eukaryotic. In some embodiments, the polynucleotides encoding the heterologous antigen are codon-optimized for expression in the eukaryotic cell. 
     A variety of expression cassettes suitable for expression of antigens in  Listeria  are provided, e.g., in U.S. Patent Publication No. 2005/0249748, incorporated by reference herein in its entirety. Additional expression cassettes suitable for expression of heterologous polypeptides in  Listeria  or mammalian cells are well known in the art. 
     A. Heterologous Polypeptides (e.g., Antigens) 
     In some embodiments, the heterologous polypeptides which are delivered or which are encoded by the nucleic acids that are delivered by the  Listeria  of the invention into cells (e.g., mammalian cells) comprise an antigen. In some embodiments, the antigen is a tumor antigen (e.g., a human tumor antigen), or an antigenic fragment or variant thereof. In some alternative embodiments, the antigen is an antigen from an infectious agent, or an antigenic fragment or variant thereof. 
     Some non-limiting examples of antigens are provided in Table 3, below. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Exemplary antigens. 
               
            
           
           
               
               
            
               
                 Antigen 
                 Reference 
               
               
                   
               
            
           
           
               
            
               
                 Tumor antigens 
               
            
           
           
               
               
            
               
                 Mesothelin 
                 GenBank Acc. No. NM_005823; U40434; NM_013404; BC003512 
               
               
                   
                 (see also, e.g., Hassan, et al. (2004) Clin. Cancer Res. 10: 3937-3942; 
               
               
                   
                 Muminova, et al. (2004) BMC Cancer 4: 19; Iacobuzio- 
               
               
                   
                 Donahue, et al. (2003) Cancer Res. 63: 8614-8622). 
               
               
                 Wilms&#39; tumor-1 
                 WT-1 isoform A (GenBank Acc. Nos. NM_000378; NP_000369). 
               
               
                 associated protein 
                 WT-1 isoform B (GenBank Acc. Nos. NM_024424; NP_077742). 
               
               
                 (Wt-1), including 
                 WT-1 isoform C (GenBank Acc. Nos. NM_024425; NP_077743). 
               
               
                 isoform A; isoform B; 
                 WT-1 isoform D (GenBank Acc. Nos. NM_024426; NP_077744). 
               
               
                 isoform C; isoform D. 
               
               
                 
                   Stratum corneum 
                 
                 GenBank Acc. No. NM_005046; NM_139277; AF332583. See 
               
               
                 chymotryptic enzyme 
                 also, e.g., Bondurant, et al. (2005) Clin. Cancer Res. 11: 3446-3454; 
               
               
                 (SCCE), and variants 
                 Santin, et al. (2004) Gynecol. Oncol. 94: 283-288; Shigemasa, et al. 
               
               
                 thereof. 
                 (2001) Int. J. Gynecol. Cancer 11: 454-461; Sepehr, et al. (2001) 
               
               
                   
                 Oncogene 20: 7368-7374. 
               
               
                 MHC class I 
                 See, e.g., Groh, et al. (2005) Proc. Natl. Acad. Sci. USA 102: 6461-6466; 
               
               
                 chain-related protein A 
                 GenBank Acc. Nos. NM_000247; BC_016929; AY750850; 
               
               
                 (MICA); MHC class I 
                 NM_005931. 
               
               
                 chain-related protein A 
               
               
                 (MICB). 
               
               
                 Gastrin and peptides 
                 Harris, et al. (2004) Cancer Res. 64: 5624-5631; Gilliam, et al. 
               
               
                 derived from gastrin; 
                 (2004) Eur. J. Surg. Oncol. 30: 536-543; Laheru and Jaffee (2005) 
               
               
                 gastrin/CCK-2 receptor 
                 Nature Reviews Cancer 5: 459-467. 
               
               
                 (also known as 
               
               
                 CCK-B). 
               
               
                 Glypican-3 (an antigen 
                 GenBank Acc. No. NM_004484. Nakatsura, et al. (2003) Biochem. 
               
               
                 of, e.g., hepatocellular 
                 Biophys. Res. Commun. 306: 16-25; Capurro, et al. (2003) 
               
               
                 carcinoma and 
                 Gasteroenterol. 125: 89-97; Nakatsura, et al. (2004) Clin. Cancer 
               
               
                 melanoma). 
                 Res. 10: 6612-6621). 
               
               
                 Coactosin-like protein. 
                 Nakatsura, et al. (2002) Eur. J. Immunol. 32: 826-836; Laheru and 
               
               
                   
                 Jaffee (2005) Nature Reviews Cancer 5: 459-467. 
               
               
                 Prostate stem cell 
                 GenBank Acc. No. AF043498; AR026974; AR302232 (see also, 
               
               
                 antigen (PSCA). 
                 e.g., Argani, et al. (2001) Cancer Res. 61: 4320-4324; Christiansen, 
               
               
                   
                 et al. (2003) Prostate 55: 9-19; Fuessel, et al. (2003) 23: 221-228). 
               
               
                 Prostate acid 
                 Small, et al. (2000) J. Clin. Oncol. 18: 3894-3903; Altwein and 
               
               
                 phosphatase (PAP); 
                 Luboldt (1999) Urol. Int. 63: 62-71; Chan, et al. (1999) Prostate 
               
               
                 prostate-specific 
                 41: 99-109; Ito, et al. (2005) Cancer 103: 242-250; Schmittgen, et al. 
               
               
                 antigen (PSA); PSM; 
                 (2003) Int. J. Cancer 107: 323-329; Millon, et al. (1999) Eur. Urol. 
               
               
                 PSMA. 
                 36: 278-285. 
               
               
                 Six-transmembrane 
                 See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442; 
               
               
                 epithelial antigen of 
                 GenBank Acc. No. NM_018234; NM_001008410; NM_182915; 
               
               
                 prostate (STEAP). 
                 NM_024636; NM_012449; BC011802. 
               
               
                 Prostate carcinoma 
                 See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442; 
               
               
                 tumor antigen-1 
                 GenBank Acc. No. L78132. 
               
               
                 (PCTA-1). 
               
               
                 Prostate 
                 See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442). 
               
               
                 tumor-inducing gene-1 
               
               
                 (PTI-1). 
               
               
                 Prostate-specific gene 
                 See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442). 
               
               
                 with homology to 
               
               
                 G protein-coupled 
               
               
                 receptor. 
               
               
                 Prostase (an antrogen 
                 See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442; 
               
               
                 regulated serine 
                 GenBank Acc. No. BC096178; BC096176; BC096175. 
               
               
                 protease). 
               
               
                 Proteinase 3. 
                 GenBank Acc. No. X55668. 
               
               
                 Cancer-testis antigens, 
                 GenBank Acc. No. NM_001327 (NY-ESO-1) (see also, e.g., Li, et 
               
               
                 e.g., NY-ESO-1; SCP- 
                 al. (2005) Clin. Cancer Res. 11: 1809-1814; Chen, et al. (2004) Proc. 
               
               
                 1; SSX-1; SSX-2; SSX- 
                 Natl. Acad. Sci. USA. 101(25): 9363-9368; Kubuschok, et al. 
               
               
                 4; GAGE, CT7; CT8; 
                 (2004) Int. J. Cancer. 109: 568-575; Scanlan, et al. (2004) Cancer 
               
               
                 CT10; MAGE-1; 
                 Immun. 4: 1; Scanlan, et al. (2002) Cancer Res. 62: 4041-4047; 
               
               
                 MAGE-2; MAGE-3; 
                 Scanlan, et al. (2000) Cancer Lett. 150: 155-164; Dalerba, et al. 
               
               
                 MAGE-4; MAGE-6; 
                 (2001) Int. J. Cancer 93: 85-90; Ries, et al. (2005) Int. J. Oncol. 
               
               
                 LAGE-1. 
                 26: 817-824. 
               
               
                 MAGE-A1, 
                 Otte, et al. (2001) Cancer Res. 61: 6682-6687; Lee, et al. (2003) 
               
               
                 MAGE-A2; 
                 Proc. Natl. Acad. Sci. USA 100: 2651-2656; Sarcevic, et al. (2003) 
               
               
                 MAGE-A3; 
                 Oncology 64: 443-449; Lin, et al. (2004) Clin. Cancer Res. 10: 5708-5716. 
               
               
                 MAGE-A4; 
               
               
                 MAGE-A6; 
               
               
                 MAGE-A9; 
               
               
                 MAGE-A10; 
               
               
                 MAGE-A12; 
               
               
                 GAGE-3/6; 
               
               
                 NT-SAR-35; BAGE; 
               
               
                 CA125. 
               
               
                 GAGE-1; GAGE-2; 
                 De Backer, et al. (1999) Cancer Res. 59: 3157-3165; Scarcella, et al. 
               
               
                 GAGE-3; GAGE-4; 
                 (1999) Clin. Cancer Res. 5: 335-341. 
               
               
                 GAGE-5; GAGE-6; 
               
               
                 GAGE-7; GAGE-8; 
               
               
                 GAGE-65; GAGE-11; 
               
               
                 GAGE-13; GAGE-7B. 
               
               
                 HIP1R; LMNA; 
                 Scanlan, et al. (2002) Cancer Res. 62: 4041-4047. 
               
               
                 KIAA1416; Seb4D; 
               
               
                 KNSL6; TRIP4; 
               
               
                 MBD2; HCAC5; 
               
               
                 MAGEA3. 
               
               
                 DAM family of genes, 
                 Fleishhauer, et al. (1998) Cancer Res. 58: 2969-2972. 
               
               
                 e.g., DAM-1; DAM-6. 
               
               
                 RCAS1. 
                 Enjoji, et al. (2004) Dig. Dis. Sci. 49: 1654-1656. 
               
               
                 RU2. 
                 Van Den Eynde, et al. (1999) J. Exp. Med. 190: 1793-1800. 
               
               
                 CAMEL. 
                 Slager, et al. (2004) J. Immunol. 172: 5095-5102; Slager, et al. 
               
               
                   
                 (2004) Cancer Gene Ther. 11: 227-236. 
               
               
                 Colon cancer associated 
                 Scanlan, et al. (2002) Cancer Res. 62: 4041-4047. 
               
               
                 antigens, e.g., 
               
               
                 NY-CO-8; NY-CO-9; 
               
               
                 NY-CO-13; 
               
               
                 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- 
                 Dosaka-Akita, et al. (2004) Clin. Cancer Res. 10: 1773-1779. 
               
               
                 tranferase V (GnT-V). 
               
               
                 Elongation factor 2 
                 Renkvist, et al. (2001) Cancer Immunol Immunother. 50: 3-15. 
               
               
                 mutated (ELF2M). 
               
               
                 HOM-MEL-40/SSX2 
                 Neumann, et al. (2004) Int. J. Cancer 112: 661-668; Scanlan, et al. 
               
               
                   
                 (2000) Cancer Lett. 150: 155-164. 
               
               
                 BRDT. 
                 Scanlan, et al. (2000) Cancer Lett. 150: 155-164. 
               
               
                 SAGE; HAGE. 
                 Sasaki, et al. (2003) Eur. J. Surg. Oncol. 29: 900-903. 
               
               
                 RAGE. 
                 See, e.g., Li, et al. (2004) Am. J. Pathol. 164: 1389-1397; Shirasawa, 
               
               
                   
                 et al. (2004) Genes to Cells 9: 165-174. 
               
               
                 MUM-1 (melanoma 
                 Gueguen, et al. (1998) J. Immunol. 160: 6188-6194; Hirose, et al. 
               
               
                 ubiquitous mutated); 
                 (2005) Int. J. Hematol. 81: 48-57; Baurain, et al. (2000) J. Immunol. 
               
               
                 MUM-2; MUM-2 Arg- 
                 164: 6057-6066; Chiari, et al. (1999) Cancer Res. 59: 5785-5792. 
               
               
                 Gly mutation; MUM-3. 
               
               
                 LDLR/FUT fusion 
                 Wang, et al. (1999) J. Exp. Med. 189: 1659-1667. 
               
               
                 protein antigen of 
               
               
                 melanoma. 
               
               
                 NY-REN series of renal 
                 Scanlan, et al. (2002) Cancer Res. 62: 4041-4047; Scanlan, et al. 
               
               
                 cancer antigens. 
                 (1999) Cancer Res. 83: 456-464. 
               
               
                 NY-BR series of breast 
                 Scanlan, et al. (2002) Cancer Res. 62: 4041-4047; Scanlan, et al. 
               
               
                 cancer antigens, e.g., 
                 (2001) Cancer Immunity 1: 4. 
               
               
                 NY-BR-62; NY- 
               
               
                 BR-75; NY-BR-85; 
               
               
                 NY-BR-62; NY-BR-85. 
               
               
                 BRCA-1; BRCA-2. 
                 Stolier, et al. (2004) Breast J. 10: 475-480; Nicoletto, et al. (2001) 
               
               
                   
                 Cancer Treat Rev. 27: 295-304. 
               
               
                 DEK/CAN fusion 
                 von Lindern, et al. (1992) Mol. Cell. Biol. 12: 1687-1697. 
               
               
                 protein. 
               
               
                 Ras, e.g., wild type ras, 
                 GenBank Acc. Nos. P01112; P01116; M54969; M54968; P01111; 
               
               
                 ras with mutations at 
                 P01112; K00654. See also, e.g., GenBank Acc. Nos. M26261; 
               
               
                 codon 12, 13, 59, or 61, 
                 M34904; K01519; K01520; BC006499; NM_006270; NM_002890; 
               
               
                 e.g., mutations G12C; 
                 NM_004985; NM_033360; NM_176795; NM_005343. 
               
               
                 G12D; G12R; G12S; 
               
               
                 G12V; G13D; A59T; 
               
               
                 Q61H. K-RAS; 
               
               
                 H-RAS; N-RAS. 
               
               
                 BRAF (an isoform of 
                 Tannapfel, et al. (2005) Am. J. Clin. Pathol. 123: 256-2601; Tsao and 
               
               
                 RAF). 
                 Sober (2005) Dermatol. Clin. 23: 323-333. 
               
               
                 Melanoma antigens, 
                 GenBank Acc. No. NM_206956; NM_206955; NM_206954; 
               
               
                 including HST-2 
                 NM_206953; NM_006115; NM_005367; NM_004988; AY148486; 
               
               
                 melanoma cell 
                 U10340; U10339; M77481. See, e g., Suzuki, et al. (1999) J. 
               
               
                 antigens. 
                 Immunol. 163: 2783-2791. 
               
               
                 Survivin 
                 GenBank Acc. No. AB028869; U75285 (see also, e.g., Tsuruma, et 
               
               
                   
                 al. (2004) J. Translational Med. 2: 19 (11 pages); Pisarev, et al. 
               
               
                   
                 (2003) Clin. Cancer Res. 9: 6523-6533; Siegel, et al. (2003) Br. J. 
               
               
                   
                 Haematol. 122: 911-914; Andersen, et al. (2002) Histol. Histopathol. 
               
               
                   
                 17: 669-675). 
               
               
                 MDM-2 
                 NM_002392; NM_006878 (see also, e.g., Mayo, et al. (1997) 
               
               
                   
                 Cancer Res. 57: 5013-5016; Demidenko and Blagosklonny (2004) 
               
               
                   
                 Cancer Res. 64: 3653-3660). 
               
               
                 Methyl-CpG-binding 
                 Muller, et al. (2003) Br. J. Cancer 89: 1934-1939; Fang, et al. (2004) 
               
               
                 proteins (MeCP2; 
                 World J. Gastreenterol. 10: 3394-3398. 
               
               
                 MBD2). 
               
               
                 NA88-A. 
                 Moreau-Aubry, et al. (2000) J. Exp. Med. 191: 1617-1624. 
               
               
                 Histone deacetylases 
                 Waltregny, et al. (2004) Eur. J. Histochem. 48: 273-290; Scanlan, et 
               
               
                 (HDAC), e.g., HDAC5. 
                 al. (2002) Cancer Res. 62: 4041-4047. 
               
               
                 Cyclophilin B (Cyp-B). 
                 Tamura, et al. (2001) Jpn. J. Cancer Res. 92: 762-767. 
               
               
                 CA 15-3; CA 27.29. 
                 Clinton, et al. (2003) Biomed. Sci. Instrum. 39: 408-414. 
               
               
                 Heat shock protein 
                 Faure, et al. (2004) Int. J. Cancer 108: 863-870. 
               
               
                 Hsp70. 
               
               
                 GAGE/PAGE family, 
                 Brinkmann, et al. (1999) Cancer Res. 59: 1445-1448. 
               
               
                 e.g., PAGE-1; PAGE-2; 
               
               
                 PAGE-3; PAGE-4; 
               
               
                 XAGE-1; XAGE-2; 
               
               
                 XAGE-3. 
               
               
                 MAGE-A, B, C, and D 
                 Lucas, et al. (2000) Int. J. Cancer 87: 55-60; Scanlan, et al. (2001) 
               
               
                 families. MAGE-B5; 
                 Cancer Immun. 1: 4. 
               
               
                 MAGE-B6; 
               
               
                 MAGE-C2; 
               
               
                 MAGE-C3; MAGE-3; 
               
               
                 MAGE-6. 
               
               
                 Kinesin 2; TATA 
                 Scanlan, et al. (2001) Cancer Immun. 30: 1-4. 
               
               
                 element modulatory 
               
               
                 factor 1; tumor protein 
               
               
                 D53; NY 
               
               
                 Alpha-fetoprotein 
                 Grimm, et al. (2000) Gastroenterol. 119: 1104-1112. 
               
               
                 (AFP) 
               
               
                 SART1; SART2; 
                 Kumamuru, et al. (2004) Int. J. Cancer 108: 686-695; Sasatomi, et 
               
               
                 SART3; ART4. 
                 al. (2002) Cancer 94: 1636-1641; Matsumoto, et al. (1998) Jpn. J. 
               
               
                   
                 Cancer Res. 89: 1292-1295; Tanaka, et al. (2000) Jpn. J. Cancer Res. 
               
               
                   
                 91: 1177-1184. 
               
               
                 Preferentially expressed 
                 Matsushita, et al. (2003) Leuk. Lymphoma 44: 439-444; Oberthuer, 
               
               
                 antigen of melanoma 
                 et al. (2004) Clin. Cancer Res. 10: 4307-4313. 
               
               
                 (PRAME). 
               
               
                 Carcinoembryonic 
                 GenBank Acc. No. M29540; E03352; X98311; M17303 (see also, 
               
               
                 antigen (CEA), 
                 e.g., Zaremba (1997) Cancer Res. 57: 4570-4577; Sarobe, et al. 
               
               
                 CAP1-6D enhancer 
                 (2004) Curr. Cancer Drug Targets 4: 443-454; Tsang, et al. (1997) 
               
               
                 agonist peptide. 
                 Clin. Cancer Res. 3: 2439-2449; Fong, et al. (2001) Proc. Natl. 
               
               
                   
                 Acad. Sci. USA 98: 8809-8814). 
               
               
                 HER-2/neu. 
                 Disis, et al. (2004) J. Clin. Immunol. 24: 571-578; Disis and Cheever 
               
               
                   
                 (1997) Adv. Cancer Res. 71: 343-371. 
               
               
                 cdk4; cdk6; p16 
                 Ghazizadeh, et al. (2005) Respiration 72: 68-73; Ericson, et al. 
               
               
                 (INK4); Rb protein. 
                 (2003) Mol. Cancer Res. 1: 654-664. 
               
               
                 TEL; AML1; 
                 Stams, et al. (2005) Clin. Cancer Res. 11: 2974-2980. 
               
               
                 TEL/AML1. 
               
               
                 Telomerase (TERT). 
                 Nair, et al. (2000) Nat. Med. 6: 1011-1017. 
               
               
                 707-AP. 
                 Takahashi, et al. (1997) Clin. Cancer Res. 3: 1363-1370. 
               
               
                 Annexin, e.g., 
                 Zimmerman, et al. (2004) Virchows Arch. 445: 368-374. 
               
               
                 Annexin II. 
               
               
                 BCR/ABL; BCR/ABL 
                 Cobaldda, et al. (2000) Blood 95: 1007-1013; Hakansson, et al. 
               
               
                 p210; BCR/ABL p190; 
                 (2004) Leukemia 18: 538-547; Schwartz, et al. (2003) Semin. 
               
               
                 CML-66; CML-28. 
                 Hematol. 40: 87-96; Lim, et al. (1999) Int. J. Mol. Med. 4: 665-667. 
               
               
                 BCL2; BLC6; 
                 Iqbal, et al. (2004) Am. J. Pathol. 165: 159-166. 
               
               
                 CD10 protein. 
               
               
                 CDC27 (this is a 
                 Wang, et al. (1999) Science 284: 1351-1354. 
               
               
                 melanoma antigen). 
               
               
                 Sperm protein 17 
                 Arora, et al. (2005) Mol. Carcinog. 42: 97-108. 
               
               
                 (SP17); 14-3-3-zeta; 
               
               
                 MEMD; KIAA0471; 
               
               
                 TC21. 
               
               
                 Tyrosinase-related 
                 GenBank Acc. No. NM_001922. (see also, e.g., Bronte, et al. 
               
               
                 proteins 1 and 2 (TRP-1 
                 (2000) Cancer Res. 60: 253-258). 
               
               
                 and TRP-2). 
               
               
                 gp100/pmel-17. 
                 GenBank Acc. Nos. AH003567; U31798; U31799; U31807; U31799 
               
               
                   
                 (see also, e.g., Bronte, et al. (2000) Cancer Res. 60: 253-258). 
               
               
                 TARP. 
                 See, e.g., Clifton, et al. (2004) Proc. Natl. Acad. Sci. USA 
               
               
                   
                 101: 10166-10171; Virok, et al. (2005) Infection Immunity 73: 1939-1946. 
               
               
                 Tyrosinase-related 
                 GenBank Acc. No. NM_001922. (see also, e.g., Bronte, et al. 
               
               
                 proteins 1 and 2 (TRP-1 
                 (2000) Cancer Res. 60: 253-258). 
               
               
                 and TRP-2). 
               
               
                 Melanocortin 1 receptor 
                 Salazar-Onfray, et al. (1997) Cancer Res. 57: 4348-4355; Reynolds, 
               
               
                 (MC1R); MAGE-3; 
                 et al. (1998) J. Immunol. 161: 6970-6976; Chang, et al. (2002) Clin. 
               
               
                 gp100; tyrosinase; 
                 Cancer Res. 8: 1021-1032. 
               
               
                 dopachrome 
               
               
                 tautomerase (TRP-2); 
               
               
                 MART-1. 
               
               
                 MUC-1; MUC-2. 
                 See, e.g., Davies, et al. (1994) Cancer Lett. 82: 179-184; Gambus, et 
               
               
                   
                 al. (1995) Int. J. Cancer 60: 146-148; McCool, et al. (1999) 
               
               
                   
                 Biochem. J. 341: 593-600. 
               
               
                 Spas-1. 
                 U.S. Published Pat. Appl. No. 20020150588 of Allison, et al. 
               
               
                 CASP-8; FLICE; 
                 Mandruzzato, et al. (1997) J. Exp. Med. 186: 785-793. 
               
               
                 MACH. 
               
               
                 CEACAM6; CAP-1. 
                 Duxbury, et al. (2004) Biochem. Biophys. Res. Commun. 317: 837-843; 
               
               
                   
                 Morse, et al. (1999) Clin. Cancer Res. 5: 1331-1338. 
               
               
                 HMGB1 (a DNA 
                 Brezniceanu, et al. (2003) FASEB J. 17: 1295-1297. 
               
               
                 binding protein and 
               
               
                 cytokine). 
               
               
                 ETV6/AML1. 
                 Codrington, et al. (2000) Br. J. Haematol. 111: 1071-1079. 
               
               
                 Mutant and wild type 
                 Clements, et al. (2003) Clin. Colorectal Cancer 3: 113-120; 
               
               
                 forms of adenomatous 
                 Gulmann, et al. (2003) Appl. Immunohistochem. Mol. Morphol. 
               
               
                 polyposis coli (APC); 
                 11: 230-237; Jungck, et al. (2004) Int. J. Colorectal. Dis. 19: 438-445; 
               
               
                 beta-catenin; c-met; 
                 Wang, et al. (2004) J. Surg. Res. 120: 242-248; Abutaily, et al. 
               
               
                 p53; E-cadherin; 
                 (2003) J. Pathol. 201: 355-362; Liang, et al. (2004) Br. J. Surg. 
               
               
                 cyclooxygenase-2 
                 91: 355-361; Shirakawa, et al. (2004) Clin. Cancer Res. 10: 4342-4348. 
               
               
                 (COX-2). 
               
               
                 Renal cell carcinoma 
                 Mulders, et al. (2003) Urol. Clin. North Am. 30: 455-465; Steffens, 
               
               
                 antigen bound by mAB 
                 et al. (1999) Anticancer Res. 19: 1197-1200. 
               
               
                 G250. 
               
            
           
           
               
            
               
                   Francisella tularensis  antigens 
               
            
           
           
               
               
            
               
                 
                   Francisella tularensis 
                 
                 Complete genome of subspecies Schu S4 (GenBank Acc. No. 
               
               
                 A and B. 
                 AJ749949); of subspecies Schu 4 (GenBank Acc. No. NC_006570). 
               
               
                   
                 Outer membrane protein (43 kDa) Bevanger, et al. (1988) J. Clin. 
               
               
                   
                 Microbiol. 27: 922-926; Porsch-Ozcurumez, et al. (2004) Clin. 
               
               
                   
                 Diagnostic. Lab. Immunol. 11: 1008-1015). Antigenic components 
               
               
                   
                 of F. tularensis include, e.g., 80 antigens, including 10 kDa and 
               
               
                   
                 60 kDa chaperonins (Havlasova, et al. (2002) Proteomics 2: 857-86), 
               
               
                   
                 nucleoside diphosphate kinase, isocitrate dehydrogenase, 
               
               
                   
                 RNA-binding protein Hfq, the chaperone ClpB (Havlasova, et al. 
               
               
                   
                 (2005) Proteomics 5: 2090-2103). See also, e.g., Oyston and Quarry 
               
               
                   
                 (2005) Antonie Van Leeuwenhoek 87: 277-281; Isherwood, et al. 
               
               
                   
                 (2005) Adv. Drug Deliv. Rev. 57: 1403-1414; Biagini, et al. (2005) 
               
               
                   
                 Anal. Bioanal. Chem. 382: 1027-1034. 
               
            
           
           
               
            
               
                 Malarial antigens 
               
            
           
           
               
               
            
               
                 Circumsporozoite 
                 See, e.g., Haddad, et al. (2004) Infection Immunity 72: 1594-1602; 
               
               
                 protein (CSP); SSP2; 
                 Hoffman, et al. (1997) Vaccine 15: 842-845; Oliveira-Ferreira and 
               
               
                 HEP17; Exp-1 
                 Daniel-Ribeiro (2001) Mem. Inst. Oswaldo Cruz, Rio de Janeiro 
               
               
                 orthologs found in 
                 96: 221-227. CSP (see, e.g., GenBank Acc. No. AB121024). SSP2 
               
               
                   P. falciparum , and 
                 (see, e.g., GenBank Acc. No. AF249739). LSA-1 (see, e.g., 
               
               
                 LSA-1. 
                 GenBank Acc. No. Z30319). 
               
               
                 Ring-infected 
                 See, e.g., Stirnadel, et al. (2000) Int. J. Epidemiol. 29: 579-586; 
               
               
                 erythrocyte survace 
                 Krzych, et al. (1995) J. Immunol. 155: 4072-4077. See also, Good, 
               
               
                 protein (RESA); 
                 et al. (2004) Immunol. Rev. 201: 254-267; Good, et al. (2004) Ann. 
               
               
                 merozoite surface 
                 Rev. Immunol. 23: 69-99. MSP2 (see, e.g., GenBank Acc. No. 
               
               
                 protein 2 (MSP2); 
                 X96399; X96397). MSP1 (see, e.g., GenBank Acc. No. X03371). 
               
               
                 Spf66; merozoite 
                 RESA (see, e.g., GenBank Acc. No. X05181; X05182). 
               
               
                 surface 
               
               
                 protein 1(MSP1); 
               
               
                 195A; BVp42. 
               
               
                 Apical membrane 
                 See, e.g., Gupta, et al. (2005) Protein Expr. Purif. 41: 186-198. 
               
               
                 antigen 1 (AMA1). 
                 AMA1 (see, e.g., GenBank Acc. No. A′13; AJ494905; AJ490565). 
               
            
           
           
               
            
               
                 Viruses and viral antigens 
               
            
           
           
               
               
            
               
                 Hepatitis A 
                 GenBank Acc. Nos., e.g., NC_001489; AY644670; X83302; 
               
               
                   
                 K02990; M14707. 
               
               
                 Hepatitis B 
                 Complete genome (see, e.g., GenBank Acc. Nos. AB214516; 
               
               
                   
                 NC_003977; AB205192; AB205191; AB205190; AJ748098; 
               
               
                   
                 AB198079; AB198078; AB198076; AB074756). 
               
               
                 Hepatitis C 
                 Complete genome (see, e.g., GenBank Acc. Nos. NC_004102; 
               
               
                   
                 AJ238800; AJ238799; AJ132997; AJ132996; AJ000009; D84263). 
               
               
                 Hepatitis D 
                 GenBank Acc. Nos, e.g. NC_001653; AB118847; AY261457. 
               
               
                 Human papillomavirus, 
                 See, e.g., Trimble, et al. (2003) Vaccine 21: 4036-4042; Kim, et al. 
               
               
                 including all 200+ 
                 (2004) Gene Ther. 11: 1011-1018; Simon, et al. (2003) Eur. J. 
               
               
                 subtypes (classed in 
                 Obstet. Gynecol. Reprod. Biol. 109: 219-223; Jung, et al. (2004) J. 
               
               
                 16 groups), such as the 
                 Microbiol. 42: 255-266; Damasus-Awatai and Freeman-Wang 
               
               
                 high risk subtypes 16, 
                 (2003) Curr. Opin. Obstet. Gynecol. 15: 473-477; Jansen and Shaw 
               
               
                 18, 30, 31, 33, 45. 
                 (2004) Annu. Rev. Med. 55: 319-331; Roden and Wu (2003) 
               
               
                   
                 Expert Rev. Vaccines 2: 495-516; de Villiers, et al. (2004) Virology 
               
               
                   
                 324: 17-24; Hussain and Paterson (2005) Cancer Immunol. 
               
               
                   
                 Immunother. 54: 577-586; Molijn, et al. (2005) J. Clin. Virol. 32 
               
               
                   
                 (Suppl. 1) S43-S51. GenBank Acc. Nos. AY686584; AY686583; 
               
               
                   
                 AY686582; NC_006169; NC_006168; NC_006164; NC_001355; 
               
               
                   
                 NC_001349; NC_005351; NC_001596). 
               
               
                 Human T-cell 
                 See, e.g., Capdepont, et al. (2005) AIDS Res. Hum. Retrovirus 
               
               
                 lymphotropic virus 
                 21: 28-42; Bhigjee, et al. (1999) AIDS Res. Hum. Restrovirus 
               
               
                 (HTLV) types I and II, 
                 15: 1229-1233; Vandamme, et al. (1998) J. Virol. 72: 4327-4340; 
               
               
                 including the 
                 Vallejo, et al. (1996) J. Acquir. Immune Defic. Syndr. Hum. 
               
               
                 HTLV type I subtypes 
                 Retrovirol. 13: 384-391. HTLV type I (see, e.g., GenBank Acc. 
               
               
                 Cosmopolitan, Central 
                 Nos. AY563954; AY563953. HTLV type II (see, e.g., GenBank 
               
               
                 African, and 
                 Acc. Nos. L03561; Y13051; AF139382). 
               
               
                 Austro-Melanesian, and 
               
               
                 the HTLV type II 
               
               
                 subtypes IIa, IIb, IIc, 
               
               
                 and IId. 
               
               
                 Coronaviridae, 
                 See, e.g., Brian and Baric (2005) Curr. Top. Microbiol. Immunol. 
               
               
                 including 
                 287: 1-30; Gonzalez, et al. (2003) Arch. Virol. 148: 2207-2235; 
               
               
                 Coronaviruses, such as 
                 Smits, et al. (2003) J. Virol. 77: 9567-9577; Jamieson, et al. (1998) 
               
               
                 SARS-coronavirus 
                 J. Infect. Dis. 178: 1263-1269 (GenBank Acc. Nos. AY348314; 
               
               
                 (SARS-CoV), and 
                 NC_004718; AY394850). 
               
               
                 Toroviruses. 
               
               
                 Rubella virus. 
                 GenBank Acc. Nos. NC_001545; AF435866. 
               
               
                 Mumps virus, including 
                 See, e.g., Orvell, etal. (2002) J. Gen. Virol. 83: 2489-2496. See, 
               
               
                 the genotypes A, C, D, 
                 e.g., GenBank Acc. Nos. AY681495; NC_002200; AY685921; 
               
               
                 G, H, and I. 
                 AF201473. 
               
               
                 Coxsackie virus A 
                 See, e.g., Brown, et al. (2003) J. Virol. 77: 8973-8984. GenBank 
               
               
                 including the serotypes 
                 Acc. Nos. AY421768; AY790926: X67706. 
               
               
                 1, 11, 13, 15, 17, 18, 
               
               
                 19, 20, 21, 22, and 24 
               
               
                 (also known as Human 
               
               
                 enterovirus C; HEV-C). 
               
               
                 Coxsackie virus B, 
                 See, e.g., Ahn, et al. (2005) J. Med. Virol. 75: 290-294; Patel, et al. 
               
               
                 including subtypes 1-6. 
                 (2004) J. Virol. Methods 120: 167-172; Rezig, et al. (2004) J. Med. 
               
               
                   
                 Virol. 72: 268-274. GenBank Acc. No. X05690. 
               
               
                 Human enteroviruses 
                 See, e.g., Oberste, et al. (2004) J. Virol. 78: 855-867. Human 
               
               
                 including, e.g., human 
                 enterovirus A (GenBank Acc. Nos. NC_001612); human 
               
               
                 enterovirus A (HEV-A, 
                 enterovirus B (NC_001472); human enterovirus C (NC_001428); 
               
               
                 CAV2 to CAV8, 
                 human enterovirus D (NC_001430). Simian enterovirus A 
               
               
                 CAV10, CAV12, 
                 (GenBank Acc. No. NC_003988). 
               
               
                 CAV14, CAV16, and 
               
               
                 EV71) and also 
               
               
                 including HEV-B 
               
               
                 (CAV9, CBV1 to 
               
               
                 CBV6, E1 to E7, E9, 
               
               
                 E11 to E21, E24 to 
               
               
                 E27, E29 to E33, and 
               
               
                 EV69 and E73), as well 
               
               
                 as HEV. 
               
               
                 Polioviruses including 
                 See, e.g., He, et al. (2003) J. Virol. 77: 4827-4835; Hahsido, et al. 
               
               
                 PV1, PV2, and PV3. 
                 (1999) Microbiol. Immunol. 43: 73-77. GenBank Acc. No. 
               
               
                   
                 AJ132961 (type 1); AY278550 (type 2); X04468 (type 3). 
               
               
                 Viral encephalitides 
                 See, e.g., Hoke (2005) Mil. Med. 170: 92-105; Estrada-Franco, et al. 
               
               
                 viruses, including 
                 (2004) Emerg. Infect. Dis. 10: 2113-2121; Das, et al. (2004) 
               
               
                 equine encephalitis, 
                 Antiviral Res. 64: 85-92; Aguilar, et al. (2004) Emerg. Infect. Dis. 
               
               
                 Venezuelan equine 
                 10: 880-888; Weaver, et al. (2004) Arch. Virol. Suppl. 18: 43-64; 
               
               
                 encephalitis (VEE) 
                 Weaver, et al. (2004) Annu. Rev. Entomol. 49: 141-174. Eastern 
               
               
                 (including subtypes IA, 
                 equine encephalitis (GenBank Acc. No. NC_003899; AY722102); 
               
               
                 IB, IC, ID, IIIC, IIID), 
                 Western equine encephalitis (NC_003908). 
               
               
                 Eastern equine 
               
               
                 encephalitis (EEE), 
               
               
                 Western equine 
               
               
                 encephalitis (WEE), 
               
               
                 St. Louis encephalitis, 
               
               
                 Murray Valley 
               
               
                 (Australian) 
               
               
                 encephalitis, Japanese 
               
               
                 encephalitis, and 
               
               
                 tick-born encephalitis. 
               
               
                 Human herpesviruses, 
                 See, e.g., Studahl, et al. (2000) Scand. J. Infect. Dis. 32: 237-248; 
               
               
                 including 
                 Padilla, et al. (2003) J. Med. Virol. 70 (Suppl. 1) S103-S110; 
               
               
                 cytomegalovirus 
                 Jainkittivong and Langlais (1998) Oral Surg. Oral Med. 85: 399-403. 
               
               
                 (CMV), Epstein-Barr 
                 GenBank Nos. NC_001806 (herpesvirus 1); NC_001798 
               
               
                 virus (EBV), human 
                 (herpesvirus 2); X04370 and NC_001348 (herpesvirus 3); 
               
               
                 herpesvirus-1 (HHV-1), 
                 NC_001345 (herpesvirus 4); NC_001347 (herpesvirus 5); X83413 
               
               
                 HHV-2, HHV-3, 
                 and NC_000898 (herpesvirus 6); NC_001716 (herpesvirus 7). 
               
               
                 HHV-4, HHV-5, 
                 Human herpesviruses types 6 and 7 (HHV-6; HHV-7) are disclosed 
               
               
                 HHV-6, HHV-7, 
                 by, e.g., Padilla, et al. (2003) J. Med. Virol. 70 (Suppl. 1)S103-S110. 
               
               
                 HHV-8, herpes B virus, 
                 Human herpesvirus 8 (HHV-8), including subtypes A-E, are 
               
               
                 herpes simplex virus 
                 disclosed in, e.g., Treurnicht, et al. (2002) J. Med. Virul. 66: 235-240. 
               
               
                 types 1 and 2 (HSV-1, 
               
               
                 HSV-2), and varicella 
               
               
                 zoster virus (VZV). 
               
               
                 HIV-1 including group 
                 See, e.g., Smith, et al. (1998) J. Med. Virol. 56: 264-268. See also, 
               
               
                 M (including subtypes 
                 e.g., GenBank Acc. Nos. DQ054367; NC_001802; AY968312; 
               
               
                 A to J) and group O 
                 DQ011180; DQ011179; DQ011178; DQ011177; AY588971; 
               
               
                 (including any 
                 AY588970; AY781127; AY781126; AY970950; AY970949; 
               
               
                 distinguishable 
                 AY970948; X61240; AJ006287; AJ508597; and AJ508596. 
               
               
                 subtypes) (HIV-2, 
               
               
                 including subtypes 
               
               
                 A-E. 
               
               
                 Epstein-Barr virus 
                 See, e.g., Peh, et al. (2002) Pathology 34: 446-450. 
               
               
                 (EBV), including 
                 Epstein-Barr virus strain B95-8 (GenBank Acc. No. V01555). 
               
               
                 subtypes A and B. 
               
               
                 Reovirus, including 
                 See, e.g., Barthold, et al. (1993) Lab. Anim. Sci. 43: 425-430; Roner, 
               
               
                 serotypes and strains 1, 
                 et al. (1995) Proc. Natl. Acad. Sci. USA 92: 12362-12366; Kedl, et 
               
               
                 2, and 3, type 1 Lang, 
                 al. (1995) J. Virol. 69: 552-559. GenBank Acc. No. K02739 
               
               
                 type 2 Jones, and type 3 
                 (sigma-3 gene surface protein). 
               
               
                 Dearing. 
               
               
                 Cytomegalovirus 
                 See, e.g., Chern, et al. (1998) J. Infect. Dis. 178: 1149-1153; Vilas 
               
               
                 (CMV) subtypes 
                 Boas, et al. (2003) J. Med. Virol. 71: 404-407; Trincado, et al. 
               
               
                 include CMV subtypes 
                 (2000) J. Med. Virol. 61: 481-487. GenBank Acc. No. X17403. 
               
               
                 I-VII. 
               
               
                 Rhinovirus, including 
                 Human rhinovirus 2 (GenBank Acc. No. X02316); Human 
               
               
                 all serotypes. 
                 rhinovirus B (GenBank Acc. No. NC_001490); Human rhinovirus 
               
               
                   
                 89 (GenBank Acc. No. NC_001617); Human rhinovirus 39 
               
               
                   
                 (GenBank Acc. No. AY751783). 
               
               
                 Adenovirus, including 
                 AY803294; NC_004001; AC_000019; AC_000018; AC_000017; 
               
               
                 all serotypes. 
                 AC_000015; AC_000008; AC_000007; AC_000006; AC_000005; 
               
               
                   
                 AY737798; AY737797; NC_003266; NC_002067; AY594256; 
               
               
                   
                 AY594254; AY875648; AJ854486; AY163756; AY594255; 
               
               
                   
                 AY594253; NC_001460; NC_001405; AY598970; AY458656; 
               
               
                   
                 AY487947; NC_001454; AF534906; AY45969; AY128640; 
               
               
                   
                 L19443; AY339865; AF532578. 
               
               
                 Varicella-zoster virus, 
                 See, e.g., Loparev, et al. (2004) J. Virol. 78: 8349-8358; Carr, et al. 
               
               
                 including strains and 
                 (2004) J. Med. Virol. 73: 131-136; Takayama and Takayama (2004) 
               
               
                 genotypes Oka, Dumas, 
                 J. Clin. Virol. 29: 113-119. 
               
               
                 European, Japanese, 
               
               
                 and Mosaic. 
               
               
                 Filoviruses, including 
                 See, e.g., Geisbert and Jahrling (1995) Virus Res. 39: 129-150; 
               
               
                 Marburg virus and 
                 Hutchinson, et al. (2001) J. Med. Virol. 65: 561-566. Marburg virus 
               
               
                 Ebola virus, and strains 
                 (see, e.g., GenBank Acc. No. NC_001608). Ebola virus (see, e.g., 
               
               
                 such as Ebola-Sudan 
                 GenBank Acc. Nos. NC_006432; AY769362; NC_002549; 
               
               
                 (EBO-S), Ebola-Zaire 
                 AF272001; AF086833). 
               
               
                 (EBO-Z), and 
               
               
                 Ebola-Reston (EBO-R). 
               
               
                 Arenaviruses, including 
                 Junin virus, segment S (GenBank Acc. No. NC_005081); Junin 
               
               
                 lymphocytic 
                 virus, segment L (GenBank Acc. No. NC_005080). 
               
               
                 choriomeningitis 
               
               
                 (LCM) virus, Lassa 
               
               
                 virus, Junin virus, and 
               
               
                 Machupo virus. 
               
               
                 Rabies virus. 
                 See, e.g., GenBank Acc. Nos. NC_001542; AY956319; AY705373; 
               
               
                   
                 AF499686; AB128149; AB085828; AB009663. 
               
               
                 Arboviruses, including 
                 Dengue virus type 1 (see, e.g., GenBank Acc. Nos. AB195673; 
               
               
                 West Nile virus, 
                 AY762084). Dengue virus type 2 (see, e.g., GenBank Acc. Nos. 
               
               
                 Dengue viruses 1 to 4, 
                 NC_001474; AY702040; AY702039; AY702037). Dengue virus 
               
               
                 Colorado tick fever 
                 type 3 (see, e.g., GenBank Acc. Nos. AY923865; AT858043). 
               
               
                 virus, Sindbis virus, 
                 Dengue virus type 4 (see, e.g., GenBank Acc. Nos. AY947539; 
               
               
                 Togaviraidae, 
                 AY947539; AF326573). Sindbis virus (see, e.g., GenBank Acc. 
               
               
                 Flaviviridae, 
                 Nos. NC_001547; AF429428; J02363; AF103728). West Nile virus 
               
               
                 Bunyaviridae, 
                 (see, e.g., GenBank Acc. Nos. NC_001563; AY603654). 
               
               
                 Reoviridae, 
               
               
                 Rhabdoviridae, 
               
               
                 Orthomyxoviridae, and 
               
               
                 the like. 
               
               
                 Poxvirus including 
                 Viriola virus (see, e.g., GenBank Acc. Nos. NC_001611; Y16780; 
               
               
                 orthopoxvirus (variola 
                 X72086; X69198). 
               
               
                 virus, monkeypox 
               
               
                 virus, vaccinia virus, 
               
               
                 cowpox virus), 
               
               
                 yatapoxvirus (tanapox 
               
               
                 virus, Yaba monkey 
               
               
                 tumor virus), 
               
               
                 parapoxvirus, and 
               
               
                 molluscipoxvirus. 
               
               
                 Yellow fever. 
                 See, e.g., GenBank Acc. No. NC_002031; AY640589; X03700. 
               
               
                 Hantaviruses, including 
                 See, e.g., Elgh, et al. (1997) J. Clin. Microbiol. 35: 1122-1130; 
               
               
                 serotypes Hantaan 
                 Sjolander, et al. (2002) Epidemiol. Infect. 128: 99-103; Zeier, et al. 
               
               
                 (HTN), Seoul (SEO), 
                 (2005) Virus Genes 30: 157-180. GenBank Acc. No. NC_005222 
               
               
                 Dobrava (DOB), Sin 
                 and NC_005219 (Hantavirus). See also, e.g., GenBank Acc. Nos. 
               
               
                 Nombre (SN), Puumala 
                 NC_005218; NC_005222; NC_005219. 
               
               
                 (PUU), and 
               
               
                 Dobrava-like Saaremaa 
               
               
                 (SAAV). 
               
               
                 Flaviviruses, including 
                 See, e.g., Mukhopadhyay, et al. (2005) Nature Rev. Microbiol. 3: 13-22. 
               
               
                 Dengue virus, Japanese 
                 GenBank Acc. Nos NC_001474 and AY702040 (Dengue). 
               
               
                 encephalitis virus, West 
                 GenBank Acc. Nos. NC_001563 and AY603654. 
               
               
                 Nile virus, and yellow 
               
               
                 fever virus. 
               
               
                 Measles virus. 
                 See, e.g., GenBank Acc. Nos. AB040874 and AY486084. 
               
               
                 Human 
                 Human parainfluenza virus 2 (see, e.g., GenBank Acc. Nos. 
               
               
                 parainfluenzaviruses 
                 AB176531; NC003443). Human parainfluenza virus 3 (see, e.g., 
               
               
                 (HPV), including HPV 
                 GenBank Acc. No. NC_001796). 
               
               
                 types 1-56. 
               
               
                 Influenza virus, 
                 Influenza nucleocapsid (see, e.g., GenBank Acc. No. AY626145). 
               
               
                 including influenza 
                 Influenza hemagglutinin (see, e.g., GenBank Acc. Nos. AY627885; 
               
               
                 virus types A, B, and C. 
                 AY555153). Influenza neuraminidase (see, e.g., GenBank Acc. 
               
               
                   
                 Nos. AY555151; AY577316). Influenza matrix protein 2 (see, e.g., 
               
               
                   
                 GenBank Acc. Nos. AY626144(. Influenza basic protein 1 (see, 
               
               
                   
                 e.g., GenBank Acc. No. AY627897). Influenza polymerase acid 
               
               
                   
                 protein (see, e.g., GenBank Acc. No. AY627896). Influenza 
               
               
                   
                 nucleoprotein (see, e.g., GenBank Acc. Nno. AY627895). 
               
               
                 Influenza A virus 
                 Hemagglutinin of H1N1 (GenBank Acc. No. S67220). Influenza A 
               
               
                 subtypes, e.g., swine 
                 virus matrix protein (GenBank Acc. No. AY700216). Influenza 
               
               
                 viruses (SIV): H1N1 
                 virus A H5H1 nucleoprotein (GenBank Acc. No. AY646426). 
               
               
                 influenza A and swine 
                 H1N1 haemagglutinin (GenBank Acc. No. D00837). See also, 
               
               
                 influenza virus. 
                 GenBank Acc. Nos. BD006058; BD006055; BD006052. See also, 
               
               
                   
                 e.g., Wentworth, et al. (1994) J. Virol. 68: 2051-2058; Wells, et al. 
               
               
                   
                 (1991) J.A.M.A. 265: 478-481. 
               
               
                 Respiratory syncytial 
                 Respiratory syncytial virus (RSV) (see, e.g., GenBank Acc. Nos. 
               
               
                 virus (RSV), including 
                 AY353550; NC_001803; NC001781). 
               
               
                 subgroup A and 
               
               
                 subgroup B. 
               
               
                 Rotaviruses, including 
                 Human rotavirus C segment 8 (GenBank Acc. No. AJ549087); 
               
               
                 human rotaviruses A to 
                 Human rotavirus G9 strain outer capsid protein (see, e.g., GenBank 
               
               
                 E, bovine rotavirus, 
                 Acc. No. DQ056300); Human rotavirus B strain non-structural 
               
               
                 rhesus monkey 
                 protein 4 (see, e.g., GenBank Acc. No. AY548957); human 
               
               
                 rotavirus, and 
                 rotavirus A strain major inner capsid protein (see, e.g., GenBank 
               
               
                 human-RVV 
                 Acc. No. AY601554). 
               
               
                 reassortments. 
               
               
                 Polyomavirus, 
                 See, e.g., Engels, et al. (2004) J. Infect. Dis. 190: 2065-2069; 
               
               
                 including simian 
                 Vilchez and Butel (2004) Clin. Microbiol. Rev. 17: 495-508; 
               
               
                 virus 40 (SV40), JC 
                 Shivapurkar, et al. (2004) Cancer Res. 64: 3757-3760; Carbone, et 
               
               
                 virus (JCV) and BK 
                 al. (2003) Oncogene 2: 5173-5180; Barbanti-Brodano, et al. (2004) 
               
               
                 virus (BKV). 
                 Virology 318: 1-9) (SV40 complete genome in, e.g., GenBank Acc. 
               
               
                   
                 Nos. NC_001669; AF168994; AY271817; AY271816; AY120890; 
               
               
                   
                 AF345344; AF332562). 
               
               
                 Coltiviruses, including 
                 Attoui, et al. (1998) J. Gen. Virol. 79: 2481-2489. Segments of 
               
               
                 Colorado tick fever 
                 Eyach virus (see, e.g., GenBank Acc. Nos. AF282475; AF282472; 
               
               
                 virus, Eyach virus. 
                 AF282473; AF282478; AF282476; NC_003707; NC_003702; 
               
               
                   
                 NC_003703; NC_003704; NC_003705; NC_003696; NC_003697; 
               
               
                   
                 NC_003698; NC_003699; NC_003701; NC_003706; NC_003700; 
               
               
                   
                 AF282471; AF282477). 
               
               
                 Calciviruses, including 
                 Snow Mountain virus (see, e.g., GenBank Acc. No. AY134748). 
               
               
                 the genogroups 
               
               
                 Norwalk, Snow 
               
               
                 Mountain group 
               
               
                 (SMA), and Saaporo. 
               
               
                 Parvoviridae, including 
                 See, e.g., Brown (2004) Dev. Biol. (Basel) 118: 71-77; Alvarez- 
               
               
                 dependovirus, 
                 Lafuente, et al. (2005) Ann. Rheum. Dis. 64: 780-782; Ziyaeyan, et 
               
               
                 parvovirus (including 
                 al. (2005) Jpn. J. Infect. Dis. 58: 95-97; Kaufman, et al. (2005) 
               
               
                 parvovirus B19), and 
                 Virology 332: 189-198. 
               
               
                 erythrovirus. 
               
               
                   
               
               
                 Each of the references, GenBank Acc. Nos., and the nucleic acids, peptides, and polypeptides cited in this table are hereby incorporated herein by reference in their entirety. 
               
            
           
         
       
     
     In some embodiments, the antigen is mesothelin, prostate stem cell antigen (PSCA), hepatitis B antigen, or hepatitis C antigen, or an antigenic fragment or variant thereof. In some embodiments, the antigen is mesothelin (e.g., human mesothelin) deleted of its signal peptide and/or GPI (glycosylphosphatidylinositol) anchor. 
     The antigenic fragment may be of any length, but is most typically at least about 6 amino acids, at least about 9 amino acids, at least about 12 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 50 amino acids, or at least about 100 amino acids. An antigenic fragment of an antigen comprises at least one epitope from the antigen. In some embodiments, the epitope is a MHC class I epitope. In other embodiments, the epitope is a MHC class II epitope. In some embodiments, the epitope is a CD4+ T-cell epitope. In other embodiments, the epitope is a CD8+ T-cell epitope. 
     A variety of algorithms and software packages useful for predicting antigenic regions (including epitopes) within proteins are available to those skilled in the art. For instance, algorthims that can be used to select epitopes that bind to MHC class I and class II molecules are publicly available. For instance, the publicly available “SYFPEITHI” algorithm can be used to predict MHC-binding peptides (Rammensee et al. (1999) Immunogenetics 50:213-9). For other examples of publicly available algorithms, see the following references: Parker et al. (1994) J. Immunol 152:163-75; Singh and Raghava (2001) Bioinformatics 17:1236-1237; Singh and Raghava (2003) Bioinformatics 19:1009-1014; Mallios (2001) Bioinformatics 17:942-8; Nielsen et al. (2004) Bioinformatics 20:1388-97; Donnes et al. (2002) BMC Bioinformatics 3:25; Bhasin, et al. (2004) Vaccine 22:3195-204; Guan et al. (2003) Nucleic Acids Res 31:3621-4; Reche et al. (2002) Hum. Immunol. 63:701-9; Schirle et al. (2001) J. Immunol Methods 257:1-16; Nussbaum et al. (2001) Immunogenetics (2001) 53:87-94; Lu et al. (2000) Cancer Res. 60:5223-7. See also, e.g., Vector NTI® Suite (Informax, Inc, Bethesda, Md.), GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.), Welling, et al. (1985) FEBS Lett. 188:215-218, Parker, et al. (1986) Biochemistry 25:5425-5432, Van Regenmortel and Pellequer (1994) Pept. Res. 7:224-228, Hopp and Woods (1981) PNAS 78:3824-3828, and Hopp (1993) Pept. Res. 6:183-190. Some of the algorthims or software packages discussed in the references listed above in this paragraph are directed to the prediction of MHC class I and/or class II binding peptides or epitopes, others to identification of proteasomal cleavage sites, and still others to prediction of antigenicity based on hydrophilicity. 
     Once a candidate antigenic fragment believed to contain at least one epitope of the desired nature has been identified, the polynucleotide sequence encoding that sequence can be incorporated into an expression cassette and introduced into a  Listeria  vaccine vector or other bacterial vaccine vector. The immunogenicity of the antigenic fragment can then be confirmed by assessing the immune response generated by the  Listeria  or other bacteria expressing the fragments. Standard immunological assays such as ELISPOT assays, Intracellular Cytokine Staining (ICS) assay, cytotoxic T-cell activity assays, or the like, can be used to verify that the fragment of the antigen chosen maintains the desired imunogenicity. In addition, the anti-tumor efficacy of the  Listeria  and/or bacterial vaccines can also be assessed using animal models (e.g., implantation of CT26 murine colon cells expressing the antigen fragment in mice, followed by vaccination of the mice with the candidate vaccine and observation of effect on tumor size, metastasis, survival, etc. relative to controls and/or the full-length antigen). 
     In addition, large databases containing epitope and/or MHC ligand information using for identifying antigenic fragments are publicly available. See, e.g., Brusic et al. (1998) Nucleic Acids Res. 26:368-371; Schonbach et al. (2002) Nucleic Acids Research 30:226-9; and Bhasin et al. (2003) Bioinformatics 19:665-666; and Rammensee et al. (1999) Immunogenetics 50:213-9. 
     The amino acid sequence of an antigenic variant has at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identity to the original antigen. 
     In some embodiments, the antigenic variant is a conservative variant that has at least about 80% identity to the original antigen and the substitutions between the sequence of the antigenic variant and the original antigen are conservative amino acid substitutions. The following substitutions are considered conservative amino acid substitutions: valine, isoleucine, or leucine are substituted for alanine; lysine, glutamine, or asparagine are substituted for arginine; glutamine, histidine, lysine, or arginine are substituted for asparagine; glutamic acid is substituted for aspartic acid; serine is substituted for cysteine; asparagine is substituted for glutamine; aspartic acid is substituted for glutamic acid; proline or alanine is substituted for glycine; asparagine, glutamine, lysine or arginine is substituted for histidine; leucine, valine, methionine, alanine, phenylalanine, or norleucine is substituted for isoleucine; norleucine, isoleucine, valine, methionine, alanine, or phenylalanine is substituted for leucine; arginine, glutamine, or asparagine is substituted for lysine; leucine, phenylalanine, or isoleucine is substituted for methionine; leucine, valine, isoleucine, alanine, or tyrosine is substituted for phenylalanine; alanine is substituted for proline; threonine is substituted for serine; serine is substituted for threonine; tyrosine or phenylalanine is substituted for tryptophan; tryptophan, phenylalanine, threonine, or serine is substituted for tyrosine; tryptophan, phenylalanine, threonine, or serine is substituted for tyrosine; isoleucine, leucine, methionine, phenylalanine, alanine, or norleucine is substituted for valine. In some embodiments, the antigenic variant is a convervative variant that has at least about 90% or at least about 95%identity to the original antigen. 
     “Percent (%) sequence identity” (or, alternatively, the “percent (%) identical”), as used herein with respect to amino acid sequences, refers to the percentage of amino acid residues in a candidate sequence (such as a variant of an antigen) that is identical to the amino acid residues in a specific reference sequence (such as in a specific antigen sequence), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions a part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using any of the publicly available algorithms and/or computer software for sequence alignment, or by inspection. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. The % sequence identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of identical matches in the optimal alignment of the A and B sequences, and where Y is the total number of amino acid residues in B. 
     Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730. 
     Alternatively, the % (amino acid) sequence identity may be obtained using one of the publicly available BLAST or BLAST-2 programs. The WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Percent (amino acid) sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The BLAST program is based on the alignment method of Karlin and Altschul. Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). 
     B. Self-Replicating RNAs and Virus-Derived Nucleic Acid Expression Cassettes 
     In some embodiments, the  Listeria  of the invention comprise a polynucleotide encoding an RNA such as a self-replicating RNA and/or a virus-derived nucleic acid expression cassette. As used herein, a “self-replicating RNA” encompasses an RNA sequence or molecule that contains all of the genetic information necessary to encode all proteins necessary for self-amplification or self-replication in an appropriate environment (e.g., in the cytosol of a mammalian host cell.) In certain embodiments, the self-replicating RNA sequences are derived from viruses, such as ssRNA positive-strand virus. The RNAs may be generated from the polynucleotide in the  Listeria  or in the cytosol of an infected cell following holin-dependent externalization. The RNAs encode a heterologous polypeptide which, in some embodiments, is expressed from the RNA within the  Listeria.  In some embodiments the heterologous polypeptide is instead translated in the cytosol of an infected cell. 
     The invention provides a polynucleotide encoding an expression cassette derived from a ssRNA positive-strand virus (no DNA stage). Also provided is a  Listeria  bacterium containing a polynucleotide, for example, a genomic or plasmid-based nucleic acid, encoding an expression cassette derived from a ssRNA positive-strand virus (no DNA stage). The expression cassette can be from, or derived from, a member of the Togaviridae; Flaviviridae; Caliciviridae; Leviviridae; Picornaviridae; Tetraviridae; Tobamovirus; Nodaviridae; Astroviridae; Barnaviridae; Nidovirales; Dicistroviridae; Iflavirus; or Hepeviridae. In some embodiments, the expression cassette is from, or derived from, a member of the Picornaviridae. For example, it can be a Enterovirus; Hepatovirus; Cardiovirus; Aphthovirus; Rhinovirus; Teschovirus; Parechovirus; Kobuvirus; or Erbovirus. Typically, the Enterovirus-derived expression cassette is from a Bovine enterovirus; Coxsackievirus; Echovirus; Porcine enterovirus B; Sheep enterovirus; Porcine enterovirus A; Human enterovirus A; Human enterovirus B; Human enterovirus C; Human enterovirus D; Poliovirus; or Simian enterovirus A. The Nidoviridae virus-derived expression cassette can be from, or derived from, Coronaviridae, Arteriviridae, or Roniviridae. Coronaviridae-derived expression cassettes have been described (see, e.g., Verheije, et al. (2006) J. Virol. 80:1250-1260; Sola, et al. (2003) J. Virol. 77:4357-4369). Sometimes, the Flaviviridae-derived expression cassette can be from a Flavivirus, Pestivirus, or Hepacivirus. In certain embodiments, the Hepatovirus-derived expression cassette is from Hepatitis A virus or Avian encephalomyelitis virus. Sometimes the Aphthovirus is Foot-and-mouth disease virus or Equine rhinitis A virus. 
     In some embodiments, the RNA generated within the  Listeria  or released from the  Listeria  comprises an expression cassette derived from an ssRNA positive-strand virus. In some embodiments, the virus is selected from the group consisting of togavirus, flavivirus, pestivirus, and picornavirus. In some embodiments, the expression cassette is derived from a togavirus. For instance, the RNA may comprise an alphavirus replicon that expresses the heterologous polypeptide. The alphavirus replicon may be derived from Sindbis virus, Venezuelan Equine Encephalitis (VEE) virus, or Semliki Forest virus (SFV). In some embodiments, the RNA comprises a flavivirus replicon (e.g., derived from the Kunjin virus) that expresses the heterologous polypeptide. Alternatively, the RNA may comprise a picornavirus replicon (e.g., a replicon derived from Encephalomycocarditis (EMCV) virus, poliovirus, or coxsackie virus) that expresses the heterologous polypeptide. For instance, the expression cassette can be derived from the Encephalomycocarditis (EMCV) virus. 
     The invention, in some aspects, provides a  Listeria  bacterium containing a polynucleotide encoding a togavirus-derived expression cassette. The polynucleotide, in some embodiments, is integrated into the listerial genome, where integration can be mediated by site-specific recombination or by homologous recombination. In some aspects, the polynucleotide is integrated by homologous recombination within a virulence factor gene, while in other aspects, the polynucleotide is not integrated within a virulence factor gene. The polynucleotide encoding the cassette is operably linked with a listerial promoter, or a synthetic promoter active in  Listeria.  In some aspects, the promoter, e.g., a prfA-dependent promoter, is specifically activated in the environment of the host cell. 
     In certain embodiments, the togavirus-derived expression cassette encodes an RNA synthesized inside the  Listeria  bacterium, where the RNA is subsequently released from the bacterium to the host cell&#39;s cytoplasm, where togavirus-encoded replication apparatus generates copies of the RNA. Using information from the expression cassette encoded RNA, the mammalian ribosome biosynthesizes non-structural viral proteins which, in turn, generate and amplify copies of the RNA. Using this information, the mammalian ribosome also synthesizes the heterologous antigens encoded by the RNA. 
     Table 4, below, discloses a number of togaviruses and genomes, contemplated for the togavirus-derived expression cassette. Togaviruses, which include alphaviruses and can include flaviviruses, have a single-stranded (+)RNA genome, where the RNA genome contains an open reading frame (ORF) encoding a polyprotein. The togavirus polyprotein contains a protease, which catalyzes cleavage of the polyprotein to generate separate non-structural proteins (nsp). The non-structural proteins of togaviruses include a protease and an RNA polymerase. The RNA polymerase catalyzes replication of the viral genome in the host cell&#39;s cytoplasm. In some embodiments, the RNA polymerase catalyzes amplification of the togavirus-derived expression cassette. 
     The togavirus-derived expression cassette of the invention is described by way of the example of alphaviruses. Alphaviruses are closely related in their genomic organization and include Sindbis virus (SIN), Semliki Forest virus, Venezualan equine encephalitis virus (VEE), Eastern equine encephalitis virus, Western equine encephalitis virus, and Ross River virus (see, e.g., Kuhn, et al. (1996) J. Virol. 70:7900-7909; Powers, et al. (2001) J. Virol. 75:10118-10131; Schlesinger (2001) Exp. Opin. Biol. Ther. 1:177-191; Frolov, et al. (1999) J. Virol. 73:3854-3865). Alphaviruses encode four non-structural proteins (nsp), nsp1, nsp2, nsp3, and nsp4. The DNA of the invention, which can be integrated into the listerial genome, encodes (+)strand RNA, which is cap-independent. The (+)strand RNA is cap-independent because at the early stages of infection, nsp1 (capping enzyme) is not yet expressed. The (+)strand RNA then encodes (−) strand RNA, where the (−) strand RNA encodes both full length genomic (+)strand RNA (which can be capped) and shorter sub-genomic (+)strand RNA, which encodes the structural proteins (or heterologous antigen) (which also can be capped). 
     After synthesis of the RNA from the alphavirus-derived expression cassette, the RNA can transit from the bacterium to the host cell&#39;s cytosol, and the RNA can be used to biosynthesize the polyprotein (P1234), where proteolytic cleavage of P1234 generates the non-structural proteins (nsp), nsp1, nsp2, nsp3, and nsp4. Nsp4 is viral RNA polymerase. 
     Togavirus genomic structure and polyproteins are described (see, e.g., (Frolov, et al. (1999) J. Virol. 73:3854-3865; Shirako, et al. (2003) J. Virol. 77:2301-2309; Vasiljeva, et al. (2003) J. Biol. Chem. 278:41636-41645; Lampio, et al. (2000) J. Biol. Chem. 275:37853-37859; Vasiljeva, et al. (2001) J. Biol. Chem. 276:30786-30793; Ackermann and Padmanabhan (2001) J. Biol. Chem 276:39926-39937; Wu, et al. (2005) J. Virol. 79:10268-10277); Amberg, et al. (1994) J. Virol. 68:3794-3802). 
     What is available are nucleic acids encoding alphavirus nsp2 mutants, where the mutation reduces possible cytotoxic effects of the alphavirus-derived expression cassette. These include mutations at amino acids 726 or 779 and at homologous positions in any homologous virus-derived genome (Frolov, et al. (1999) J. Virol. 73:3854-3865). 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Sources of alphavirus- and flavivirus-derived expression cassettes. 
               
               
                   
               
             
            
               
                 Alphaviruses 
               
            
           
           
               
               
            
               
                 Sindbis virus 
                 Dubensky, et al. (1996) J. Virol. 70: 508-519; Perri, et al. 
               
               
                   
                 (2003) J. Virol. 77: 10394-10403; Lindenbach and Rice (1997) 
               
               
                   
                 J. Virol. 71: 9608-9617; Perri, et al. (2000) J. Virol. 74: 9802-9807. 
               
               
                   
                 See, e.g., GenBank Acc. No. NC_001547. 
               
               
                 Sindbis virus strain 
                 Smith and Tignor (1980) Arch. Virol. 66: 11-26; Yu, et al. 
               
               
                 SAAR86 
                 (1998) J. Biol. Chem. 273: 23524-23533; GenBank Acc. Nos. 
               
               
                   
                 AF061686 and AF061207. 
               
               
                 Semliki Forest 
                 Nordstrom, et al. (2005) J. Gen. Virol. 86: 349-354; Tannis, et 
               
               
                   
                 al. (2005) Vaccine 23: 4189-4194; Diatta, et al. (2005) J. Gen. 
               
               
                   
                 Virol. 86: 3129-3136; Karlsson and Liljestrom (2004) Methods 
               
               
                   
                 Mol. Biol. 246: 543-557; Perri, et al. (2000) J. Virol. 74: 9802-9807; 
               
               
                 Venezuelan equine 
                 Perri, et al. (2000) J. Virol. 74: 9802-9807; Perri, et al. (2003) 
               
               
                 encephalitis (VEE) 
                 J. Virol. 77: 10394-10403; Balasuriya, et al. (2000) J. Virol. 
               
               
                   
                 74: 10623-10630; Gehrke, et al. (2005) J. Gen. Virol. 86: 1045-1053; 
               
               
                   
                 Cassetti, et al. (2004) Vaccine 22: 520-527; Thompson, 
               
               
                   
                 et al. (2006) Proc. Natl. Acad. Sci. USA 103: 3722-3727; 
               
               
                 Eastern equine 
                 See, e.g., Petrakova, et al. (2005) J. Virol. 79: 7597-7608. 
               
               
                 encephalitis (EEE) 
                 GenBank Acc. Nos. AY705240; AY722102; AY705241. 
               
               
                 Western equine 
                 See, e.g., GenBank Acc. No. NC_003908. 
               
               
                 encephalitis 
               
               
                 Ross river virus 
                 See, e.g., Frolov, te al. (1997) J. Virol. 71: 2819-2829; Frolova, 
               
               
                   
                 et al. (1997) J. Virol. 71: 248-258; Faragher, et al. (1988) 
               
               
                   
                 Virology 163: 509-526; GenBank Acc. No. NC_001544. 
               
               
                 Sagiyami virus. 
                 See, e.g., Shirako and Yamaguchi (2000) J. Gen. Virol. 
               
               
                   
                 81: 1353-1360. 
               
               
                 O&#39;Nyong-nyong virus 
                 GenBank Acc. Nos. NC_001512; AF079456; M20303. 
               
               
                   
                 Myles, et al. (2006) J. Virol. 49: 4992-4997. 
               
               
                 Highlands J virus 
                 GenBank Acc. Nos. AF023289; J02206; K00700; AH002349. 
               
               
                   
                 Bianchi, et al. (1993) Am. J. Trop. Med. Hyg. 49: 322-328. 
               
            
           
           
               
            
               
                 Flaviviruses 
               
            
           
           
               
               
            
               
                 Yellow fever 
                 Jones, et al. (2005) Virology 331: 247-259; Molenkamp, et al. 
               
               
                   
                 (2003) J. Virol. 77: 1644-1648. 
               
               
                 Yellow fever strain 17D 
                 Lindenbach and Rice (1997) J. Virol. 71: 9608-9617; 
               
               
                   
                 Barba-Spaeth, et al. (2005) J. Exp. Med. 202: 1179-1184; 
               
               
                   
                 Pugachev, et al. (2005) Curr. Opin. Infect. Dis. 18: 387-394; 
               
               
                   
                 Bonaldo, et al. (2005) J. Virol. 79: 8602-8613; Bredenbeek, et 
               
               
                   
                 al. (2006) Virology 345: 299-304. See also, e.g., GenBank 
               
               
                   
                 Acc. No. X03700. 
               
               
                 Japanese encephalitis 
                 See, e.g., GenBank Acc. Nos. AB24119; AB24118; 
               
               
                   
                 AB196926. 
               
               
                 St. Louis encephalitis 
                 See, e.g., GenBank Acc. No. NC_007580. 
               
               
                 Tick-borne encephalitis 
                 Aberle, et al. (2005) J. Virol. 79: 15107-15113; Gehrke, et al. 
               
               
                   
                 (2005) J. Gen. Virol. 86: 1045-1053. See, e.g., GenBank Acc. 
               
               
                   
                 No. AF069066. 
               
               
                 Dengue virus 
                 Medlin, et al. (2005) J. Virol. 79: 11053-11061; Alvarez, et al. 
               
               
                   
                 (2005) Virology 339: 200-212; Pang, et al. (2001) 1: 28; 
               
               
                   
                 Aberle, et al. (2005) J. Virol. 79: 15107-15113; 
               
               
                 West Nile virus 
                 Aberle, et al. (2005) J. Virol. 79: 15107-15113; Fayzulin, et al. 
               
               
                   
                 (2006) Virology April 26 [epub ahead of print]. See, e.g., 
               
               
                   
                 GenBank Acc. Nos. DQ411034; DQ411033. 
               
               
                 Kunjin virus (subtype of 
                 Tannis, et al. (2005) Vaccine 23: 4189-4194; Anraku, et al. 
               
               
                 West Nile virus) 
                 (2002) J. Virol. 76: 3791-3799; Liu, et al. (2004) J. Virol. 
               
               
                   
                 78: 1225-12235; 
               
            
           
           
               
            
               
                 Arterivirus 
               
            
           
           
               
               
            
               
                 Equine arteritis virus 
                 Pasternak, et al. (2004) J. Virol. 78: 8102-8113. See, e.g., 
               
               
                   
                 GenBank Acc. Nos. NC_002532; AY349168. 
               
            
           
           
               
            
               
                 Rubivirus 
               
            
           
           
               
               
            
               
                 Rubella virus 
                 Tzeng, et al. (2005) J. Clin. Microbiol. 43: 879-885; Chen and 
               
               
                   
                 Icenogle (2004) J. Virol. 78: 4314-4322; Tzeng and Frey 
               
               
                   
                 (2005) Virology 337: 327-334. See, e.g., GenBank Acc. Nos. 
               
               
                   
                 AF435866; NC_001545. 
               
               
                   
               
               
                 The listerial genome of the invention encompasses a  Listeria -compatible transcription start sequence operably linked with a togavirus-derived expression cassette, where transcription in the bacterium produces an RNA, and where the RNA comprises a mammal-compatible transcription start sequence operably linked with at least one open reading frame (ORF), and where the ORF includes at least one nucleic acid encoding a heterologous antigen. 
               
            
           
         
       
     
     Togavirus-derived expression cassettes, including alphavirus-derived expression cassettes, have been described (See, e.g., Dubensky, et al. (1996) J. Virol. 70:508-519 and U.S. Pat. No. 6,342,372 of Dubensky, et al.). Reagents and methods relating to alphavirus-derived vectors, and to yellow fever virus (a flavivirus)-derived vectors are disclosed. Alphaviruses-based vectors are available (see, e.g., U.S. Pat. No. 5,789,245 issued to Dubensky, et al.; U.S. Pat. No. 5,814,482 issued to Dubensky, et al.; U.S. Pat. No. 5,843,723 issued to Dubensky, et al.; U.S. Pat. No. 6,015,686 issued to Dubensky, et al.; U.S. Pat. No. 6,426,196 issued to Dubensky, et al.; U.S. Pat. No. 6,451,592 issued to Dubensky, et al.; U.S. Pat. No. 6,458,560 issued to Dubensky, et al.; and U.S. Pat. No. 6,465,634 issued to Dubensky, et al.). Yellow fever virus-derived vectors are available (see, e.g., U.S. Pat. No. 6,696,281 issued to Chambers, et al.; U.S. Pat. No. 6,962,708 issued to Chambers, et al., U.S. Pat. No. 5,744,141 issued to Paoletti and Pincus; Bonaldo, et al. (2005) J. Virol. 79:8602-8613; Bredenbeek, et al. (2006) Virology 345:299-304; McAllister, et al. (2000) J. Virol. 74:9197-9295; Tao, et al. (2005) J. Immunol. 201:201-209). 
     In certain embodiments, the nucleic acid encoding holin and the togavirus-derived expression cassette reside in the same bacterium. But the nucleic acid encoding holin and the togavirus-derived expression cassette need not be supplied by the same bacterium. Rather, they can be provided by two different vectors. Where a first  Listeria  bacterium provides the togavirus-derived expression cassette, a second  Listeria  bacterium can provide a nucleic acid encoding holin. In another aspect, the holin can be supplied by a naked nucleic acid vector, adenovirus-derived vector, and so on. What is also provided is a dendric cell (DC) vaccine, where the DC is infected in vitro with the  Listeria  containing the togavirus-derived cassette and/or the holin (see, e.g., WO 2005/009463). 
     In other aspects, the invention provides a  Listeria  bacterium containing an alphavirus-derived expression cassette derived from an alphavirus that tends to stimulate greater interferon response against the alphavirus, such as Sindbis virus, as well as an alphavirus-derived expression cassette derived from an alphavirus that stimulates lesser interferon responses against the alphavirus, such as VEE or yellow fever virus. 
     C. Cis-Acting RNA Elements Used in Replication 
     A cis-acting RNA element for stimulating replication is utilized in some embodiments of the invention, where this element requires nucleotides 5′-prime to the open reading frame encoding non-structural protein-1 (nsp1) and also requires a number of nucleotides within the ORF for nsp1. The cis-acting element overlaps the start codon of nsp1, and encompasses nucleotides both upstream and downstream of this start codon. 
     In certain embodiments where the invention provides for an IRES, for use in initiating translation of nsp1, the IRES is implanted just upstream of the nsp1 ORF, necessitating disruption of the cis-acting RNA element. The invention thus provides a construct that contains the cis-acting RNA element, as well as the IRES, while avoiding disruption of any part of the cis-acting RNA element. Disruption is avoided by providing an RNA containing the following nucleic acids in the following order, from 5′-prime to 3′-prime direction: First nucleic acid: Complete, intact cis-acting RNA element, where the cis-acting RNA element (in one embodiment) includes at least the part of nsp1 that is necessary for cis-acting RNA element activity; Second nucleic acid: IRES; and Third nucleic acid: Open reading frame for entire nsp1 (Table 5). As is evident from the order of the first, second, and third nucleic acids, part of the nsp1 sequence is duplicated. A potential problem in duplicate regions is the generation of artefacts within the bacterium, where the artefacts are produced by homologous recombination. The invention provides for preventing these artefacts as follows. Regarding the two duplicate regions, the nucleotide sequence of the second duplicate region is changed so that it is no longer homologous to the first duplicate region, while not changing the amino acids that are encoded by the second duplicate region. 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
             
            
               
                 Alphavirus based expression cassette, and components thereof. 
               
            
           
           
               
               
               
            
               
                 Wild type non- 
                   
                   
               
               
                 structural protein 
               
               
                 (nspt) sequence. 
                 GenBank Acc. No. NP_062889). 
               
               
                   
               
               
                 IRES sequence, and 
                 Gagctcgtatggacatattgtcgttagaacgcggctacaattaatacat 
               
               
                 sequences upstream 
                 AaccttatgtatcatacacatacgatttaggggacactatagGGATATA 
               
               
                 and downstream to the 
                 GTGGTGAGTATCCCCGCCTGTCACGCGGGAGACCGGGGTTCGGTTCCCC 
               
               
                 IRES sequence. 
                 GACGGGGAGCcaaacagccgaccaattgcactaccatcacaatggagaa 
               
               
                 (SEQ ID NO: 11) 
                 Gccagtagtaaacgtagacgtagacccccagagtccgtttgtcgtgcaa 
               
               
                   
                 Ctgcaaaaaagcttcccgcaatttgaggtagtagcacagcaggtcactc 
               
               
                   
                 CaaatgaccatgctaatgccagagcattttcgcatctggcGCATGCATC 
               
               
                   
                 TAGGGCGGCCAATTCCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTG 
               
               
                   
                 GCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTGATT 
               
               
                   
                 TTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGC 
               
               
                   
                 CCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAG 
               
               
                   
                 GAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGC 
               
               
                   
                 TTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAAC 
               
               
                   
                 CCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAG 
               
               
                   
                 ATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGAT 
               
               
                   
                 AGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGG 
               
               
                   
                 CTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGC 
               
               
                   
                 CTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCT 
               
               
                   
                 AGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGA 
               
               
                   
                 TAAGCTTatggaaaaaccggtggtcaatgtggatgtcgatccacaaagc 
               
               
                   
                 Ccattcgtagtacagcttcagaagtcatttccacagttcgaagtggtcg 
               
               
                   
                 Cccagcaagtaaccccgaacgaccacgccaacgcaagagccttcagcca 
               
               
                   
                 Cctggcc 
               
               
                   
               
               
                 Sp6 promoter. This 
                 atttaggggacactatag 
               
               
                 promoter allows 
               
               
                 transcription of 
               
               
                 downstream material, 
               
               
                 including the 
               
               
                 DI sequence, a first 
               
               
                 nsp1 sequence (nt 
               
               
                 corresponding to only 
               
               
                 about the first 48 
               
               
                 amino acids of nsp1), 
               
               
                 the IRES sequence, 
               
               
                 and a second nsp1 
               
               
                 (full-length nsp1 
               
               
                 sequence). 
               
               
                 SEQ ID NO: 12) 
               
               
                   
               
               
                 “DI sequence.” This 
                 GGATATAGTGGTGAGTATCCCCGCCTGTCACGCGGGAGACCGGGG 
               
               
                 sequence, which 
                 TTCGGTTCCCCGACGGGGAGC 
               
               
                 resembles tRNA ASP , 
               
               
                 enhances stability of 
               
               
                 the expressed 
               
               
                 message. 
               
               
                 (SEQ ID NO: 13) 
               
               
                   
               
               
                 Sequence that 
                 Caaacagccgaccaattgcactaccatcacaatgga 
               
               
                 enhances replication. 
                 GaagccagtagtaaacgtagacgTagacccccagag 
               
               
                 (SEQ ID NO: 14) 
                 Tccgtttgtcgtgcaactgcaaaaaagcttcccgca 
               
               
                   
                 Atttgaggtagtagcacagcaggtcactccaaatga 
               
               
                   
                 Ccatgctaatgccagagcattttcgcatctggc 
               
               
                   
               
            
           
           
               
               
            
               
                 The sequence that enhances replication has two regions: (1) The first 
                   
               
               
                 region is upstream of the nsp1-encoding region: 
               
               
                 Caaacagccgaccaattgcactaccatcaca; and (2) The second region 
               
               
                 is a fragment of the nsp1-encoding region: atg gaG aag cca gta 
               
               
                 gta aac gta gac gTa gac ccc cag agT ccg ttt gtc gtg caa ctg caa aaa 
               
               
                 agc ttc ccg caA ttt gag gta gta gca cag cag gtc act cca aat gaC cat gct 
               
               
                 aat gcc aga gca ttt tcg cat ctg gc (SEQ ID NO: 15). 
               
               
                   
               
            
           
           
               
               
               
            
               
                 ECVM internal ribosome 
                 GCATGCATCTAGGGCGGCCAATTCCGCCCCTCTCCCTCCCCCCCCCC 
                   
               
               
                 entry sequence 
                 TAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTG 
               
               
                 (IRES) sequence. 
                 TCTATATGTGATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGG 
               
               
                 (SEQ ID NO: 16) 
                 GCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCT 
               
               
                   
                 TTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGG 
               
               
                   
                 AAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCG 
               
               
                   
                 ACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTG 
               
               
                   
                 CGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAAC 
               
               
                   
                 CCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGG 
               
               
                   
                 CTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGT 
               
               
                   
                 ACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA 
               
               
                   
                 CATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCAC 
               
               
                   
                 GGGGACGTGGTTTTCCTTTGAAAAACACGATGATAAGCTT 
               
               
                   
               
               
                 First 50 codons of the 
                 Atggaaaaaccggtggtcaatgtggatgtcgatccacaa 
               
               
                 second nsp1 sequence. 
                 Agcccattcgtagtacagcttcagaagtcatttccacag 
               
               
                 These nucleotides 
                 Ttcgaagtggtcgcccagcaagtaaccccgaacgaccac 
               
               
                 (nucleotides altered) 
                 gccaacgcaagagccttcagccacctg 
               
               
                 correspond to the first 
               
               
                 nsp1 sequence 
               
               
                 (nucleotides not 
               
               
                 altered). 
               
               
                 To prevent generation 
               
               
                 of artifacts in vivo, 
               
               
                 resulting from 
               
               
                 homologous 
               
               
                 recombination, a 
               
               
                 number of nucleotides 
               
               
                 were changed in the 
               
               
                 second nsp1 sequence 
               
               
                 (without changing the 
               
               
                 encoded amino acids 
               
               
                 that were encoded by 
               
               
                 the second nsp1 
               
               
                 sequence). 
               
               
                 (SEQ ID NO: 17) 
               
               
                   
               
            
           
         
       
     
     D. Internal Ribosome Entry Sites (IRES). 
     An internal ribosome entry site (IRES), useful for operably linking with a nucleic acid encoding a heterologous antigen, is available for the invention. For example, the invention encompasses a  Listeria  bacterium containing a togavirus-derived expression cassette, where the expression cassette contains at least one IRES, and where the IRES is operably linked with a nucleic acid encoding a heterologous antigen. 
     IRES sequences, also called cap-independent translation enhancers (CITE), are stretches of about 400-500 ribonucleotides residing either at the 5′-prime end of mRNA or at internal sites in the mRNA. The IRES sequence is used to initiate translation. In detail, the IRES sequence can mediate entry of a ribosome and initiate translation at an internal site of an mRNA that lacks a cap (see, e.g., Jimenez, et al. (2005) RNA 11:1385-1399; Lytle, et al. (2001) J. Virol. 75:7629-7636; Boni, et al. (2005) J. Biol. Chem. 280:17737-17748; Belsham and Sonenberg (1996) Microbiol. Revs. 60:499-551; Makrides (1999) Protein Expression and Purification 17:183-202; Borman, et al. (1997) Nucl. Acids Res. 25:925-932; Mountford and Smith (1995) Trends Genet. 11:179-184). IRES sequences can be useful in the following situation. The eukaryotic translation machinery sometimes cannot use the second ORF of a bicistronic message. However, where an IRES resides upstream (5′-prime) to the second open reading frame, the eukaryotic translation machinery readily uses the second ORF for polypeptide synthesis. 
     IRES sequences available for the reagents and methods of the invention include, but are not limited to, IRES sequences from hepatoviruses (e.g., hepatitis A virus; hepatitis C virus), cardioviruses (e.g., encephalomyocarditis virus; mengovirus; Theiler&#39;s murine encephalomyelitis virus; echovirus 22), aphthaoviruses (e.g., foot and mouth disease virus (FMDV)), rhinoviruses, and enteroviruses (e.g., polioviruses; coxsackie A21 virus; enterovirus 70; coxsackie B virus; coxsackie A9 viruses; coxsackie A16 virus; echoviruses, and bovine enterovirus). IRES sequences occur in pestiviruses and GB virus B, picornaviruses, simian immunodeficiency virus (SIV), retro-elements such as VL-30, and retroviruses (e.g., Friend murine leukemia virus; Moloney murine leukemia virus (MMLV); human T-cell leukemia virus; reticuloendotheliosis virus type A). A number of IRES sequences have also been identified in mRNAs encoding mammalian proteins (“cellular IRES”) (see, e.g., Chappell and Mauro (2003) J. Biol. Chem. 278:33793-33800; Bornes, et al. (2004) J. Biol. Chem. 279:18717-18726; Ali, et al. (2000) J. Biol. Chem. 275:27531-27540; Komar and Hatzoglou (2005) J. Biol. Chem. 280:23425-23428; Jackson and Kaminski (1995) RNA 1:985-1000; Fernandez, et al. (2001) J. Biol. Chem. 276:12285-12291; Ohlmann, et al. (2000) J. Biol. Chem. 275:11899-11906; Sachs (2000) Cell 101:243-245; Stoneley and Willis (2004) Oncogene 23:3200-3207). IRES elements from one or more of HRV (110 to 640, numbered from 5′-end of viral genome), FMDV (-445 to 1, numbered from initiation codon), HAV (225 to 746, numbered from 5′-end of genome), HCV (40 to 380, numbered from 5′-end of genome), GBV-B (61 to 460), GBV-C (60 to 690), CSFV (65-376, numbered from 5′-end of genome), and HHV8 (−225 to 1, numbered from initiation codon), are available for use in the present invention (see, e.g., Beales, et al. (2003) J. Virol. 77:6574-6579). 
     Table 6 discloses a number of IRES sequences, available for use in the invention, e.g., where the IRES can be integrated near or at the 5′-prime end of a togavirus-derived expression cassette, at an internal position in the togavirus-derived expression cassette, and where the IRES is operably linked with a nucleic acid encoding an open reading frame (ORF). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Internal ribosome entry site (IRES) sequences. 
               
            
           
           
               
               
            
               
                 Source 
                 Sequence 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Encephalomyocarditis 
                 Tcccccccccctaacgttactggccgaagccgcttggaataaggccg 
                   
               
               
                 virus 
                 Gtgtgcgtttgtctatatgttattttccaccatattgccgtcttttg 
               
               
                 strain HB1 IRES 
                 Gcaatgtgagggcccggaaacctggccctgtcttcttgacgagcatt 
               
               
                 (nt 167-745 
                 Cctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaa 
               
               
                 of GenBank Acc. No. 
                 Tgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaa 
               
               
                 DQ464063). See 
                 Cgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgac 
               
               
                 also, nt 302-880 of 
                 Aggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaa 
               
               
                 GenBank Acc. No. 
                 Ggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaa 
               
               
                 X74312; and 
                 Gagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggat 
               
               
                 Kaminski and 
                 Gcccagaaggtaccccattgtatgggatctgatctggggcctcggtg 
               
               
                 Jackson (1998) RNA 
                 Cacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccc 
               
               
                 4: 626-638). 
                 Ccctaaccacggggacgtggttttcctttgaaaaacacgatgataat 
               
               
                 (SEQ ID NO: 18) 
                 Atggccacaaccatggaacaagagac 
               
               
                   
               
               
                 Hepatitis C 
                 Agaccacaacggtttccctctagcgggatcaattccgcccctctccc 
               
               
                 Virus IRES 
                 Tcccccccccctaacgttactggccgaagccgcttggaataaggccg 
               
               
                 (GenBank Acc. No. 
                 Gtgtgcgtttgtctatatgttattttccaccatattgccgtcttttg 
               
               
                 AJ242653) (nt 
                 Gcaatgtgagggcccggaaacctggccctgtcttcttgacgagcatt 
               
               
                 1202-1812) 
                 Cctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaa 
               
               
                 (SEQ ID NO: 19) 
                 Tgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaa 
               
               
                   
                 Cgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgac 
               
               
                   
                 Aggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaa 
               
               
                   
                 Ggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaa 
               
               
                   
                 Gagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggat 
               
               
                   
                 Gcccagaaggtaccccattgtatgggatctgatctggggcctcggtg 
               
               
                   
                 Cacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccc 
               
               
                   
                 cccgaaccacggggacgtggttttcctttgaaaaacacgataatacc 
               
               
                   
               
               
                 Foot and mouth 
                 Ggtttccacaactgataaaactcgtgcaacttgaaactccgcctggt 
               
               
                 disease virus O IRES 
                 Ctttccaggtctagaggggttacactttgtactgtgctcgactccac 
               
               
                 GenBank Acc. No. 
                 Gcccggtccactggcgggtgttagtagcagcactgttgtttcgtagc 
               
               
                 NC004004) 
                 Ggagcatggtggccgtgggaactcctccttggtgacaagggcccacg 
               
               
                 (nt 600-1058). 
                 Gggccgaaagccacgtccagacggacccaccatgtgtgcaaccccag 
               
               
                 (SEQ ID NO: 20) 
                 Cacggcaacttttactgcgaacaccaccttaaggtgacactggtact 
               
               
                   
                 Ggtactcggtcactggtgacaggctaaggatgcccttcaggtacccc 
               
               
                   
                 Gaggtaacacgggacactcgggatctgagaaggggattgggacttct 
               
               
                   
                 Ttaaaagtgcccagtttaaaaagcttctacgcctgaataggcgaccg 
               
               
                   
                 gaggccggcgcctttccattacccacta ctaaatcc 
               
               
                   
               
               
                 Kunjin virus IRES. 
                 Khromykh and Westaway (1997) J. Virol. 71: 1497-1505. (Kunjin is in 
               
               
                   
                 Flaviviridae family.) 
               
               
                   
               
               
                 Encephalomyocarditis 
                 Hoffman and Palmenberg (1995) J. Virol. 69: 4399-4406; Pugachev, et al. 
               
               
                 virus (EMCV) IRES. 
                 (2000) J. Virol. 74: 10811-10815. (Rubella virus is in Togaviridae 
               
               
                   
                 family.) 
               
               
                   
               
               
                 GB virus B (GBV-B) 
                 Rijnbrand, et al. (2000) J. Virol. 74: 773-783. (GB virus is in 
               
               
                 IRES. 
                 Flaviviridae family). GB virus B IRES has no specific requirement 
               
               
                   
                 for polyprotein sequences (Rijnbrand, et al., supra). 
               
               
                   
               
               
                 Foot-and-mouth 
                 Meyer, et al. (1995) J. Virol. 69: 2819-2824. 
               
               
                 disease virus IRES. 
               
               
                   
               
               
                 Echovirus IRES. 
                 Bradrick, et al. (2001) J. Virol. 75: 6472-6481. Echovirus IRES 
               
               
                   
                 sequences is functional with engineered into coxsackievirus (Bradrick, et 
               
               
                   
                 al., supra). 
               
               
                   
               
               
                 Hepatitis C virus 
                 Jubin, et al. (2000) J. Virol. 74: 10430-10437. 
               
               
                 IRES. 
               
               
                   
               
               
                 Swine fever virus 
                 Fletcher and Jackson (2002) J. Virol. 76: 5024-5033. 
               
               
                 (CSFV) IRES. 
               
               
                   
               
               
                 Lymphoid enhancer 
                 Jimenez, et al. (2005) RNA 11: 1385-1399. 
               
               
                 factor-1 IRES. 
               
               
                   
               
               
                 c-myc IRES. 
                 Nanbru, et al. (1997) J. Biol. Chem. 272: 32061-32066. 
               
               
                   
               
               
                 FGF-2 IRES. 
                 Nanbru, et al. (1997) J. Biol. Chem. 272: 32061-32066. 
               
               
                   
               
            
           
         
       
     
     E. Stabilizing Nucleic Acids Useful in Virus-Derived Expression Cassettes (e,g., Togavirus-Derived  Expression Cassettes). 
     Nucleic acids that maintain or enhance stability of the RNA expressed from a virus-derived expression cassette (including, but not limited to, togavirus-derived expression cassette), or enhance stability of message amplified from the RNA, are available. Stabilizing nucleic acids includes those residing at or near the 5′-prime end of an expressed RNA. Suitable stabilizing nucleic acids include, but are not limited to, tRNA-like structures and structures derived from tRNA (see, e.g., Agapov, et al. (1998) Proc. Natl. Acad. Sci. USA 95:12989-12994; Monroe and Schlesinger (1983) Proc. Natl. Acad. Sci. USA 80:3279-3283). 
     Table 7 discloses a number of useful stabilizing structures. The effect of the stabilizing nucleic acid can be measured, or inferred from, the intracellular concentration of the RNA, size range of the RNA, expression of a polypeptide from the RNA, immune response to a polypeptide from the RNA, and the like. 
     
       
         
           
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Nucleic acids encoding a stabilizing structure, useful at or 
                   
               
               
                 near the 5′-prime end of a virus-derived expression cassette 
               
               
                 (e,g., togavirus-derived expression cassette). 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Sequence resembling 
                 Atatagtggtgagtatccccgcctgtcacgcgggagac 
                   
               
               
                 tRNA Asp  identified in an 
                 cggggttcggttccccgacggggagcca 
               
               
                 alphaviral Defective 
               
               
                 Interfering (DI) particle 
               
               
                 (Monroe and 
               
               
                 Schlesinger (1983) 
               
               
                 Proc. Natl. Acad. Sci. 
               
               
                 USA 80: 3279-3283). 
               
               
                 (SEQ ID NO: 21) 
               
               
                   
               
               
                 tRNA Asp  (Sekiya, et al. 
                 tcctcgttagtatagtggtgagtatccccgcctgtcac 
               
               
                 (1981) Nucleic Acids 
                 gcgggagaccggggttcgattccccgacggggag 
               
               
                 Res. 9: 2239-2250; 
               
               
                 GenBank Acc. No. 
               
               
                 V01272) 
               
               
                 (SEQ ID NO: 22) 
               
               
                   
               
            
           
         
       
     
     Available stabilizing nucleic acids include sequences that bind an RNA-binding protein, for example, La protein and iron response element (IRE) (see, e.g., Heise, et al. (2001) J. Virol. 75:6874-6883). Another available stabilizing structure is the 5′-UTR of a stable mammalian mRNA, such as that for β-globin (Makrides (1999) Protein Expression Purification 17:183-202; Hedley, et al. (1998) Hum. Gene Ther. 9:325-332; Strong, et al. (1997) Gene Ther. 4:624-627). Increased stability can also be provided, in the invention, by including, in the construct, a nucleic acid encoding a non-togavirus capping enzyme (see, e.g., Ahola and Kaariainen (1995) Proc. Natl. Acad. Sci. USA 92:507-511; Vasiljeva, et al. (2000) J. Biol. Chem. 275:17281-17287). Stabilizing nucleic acids also encompass using a togavirus-derived expression cassette with potential RNase cleavage sites removed. Other stabilizing nucleic acids are disclosed (see, e.g., Arnold, et al. (1998) RNA 4:319-330; Bouvet, et al. (1992) Nature 360:488-491; Heck, et al. (1996) Mol. Microbiol. 20:1165-1178; Matsunaga, et al. (1996) RNA 2:1228-1240). 
     Codon optimization can be applied to the nucleic acid encoding the togavirus-derived expression cassette. Codon optimization can be applied to the proteins of viral origin, as well as to the heterologous antigen (e.g., tumor antigens; hepatitis virus antigens) proteins. Although a variety of viruses infect human cells, and although most tumor antigens are human antigens, what is contemplated, in some embodiments, is improved polypeptide biosynthesis accomplished by codon optimization for expression in human cells. Guidance in codon optimization, for example, using a human consensus codon usage table, for expresson in human cells is available (see, e.g., Ivory and Chadee (2004) Genetic Vaccines and Therapy 2:17-25; Makrides (1999) Protein Expression Purification 17:183-202; Ko, et al. (2005) Infection Immunity 73:5666-5674). 
     IV.  Listeria    
     In some embodiments, the  Listeria  belong to the species  Listeria monocytogenes.  In some alternative embodiments the bacteria are members of the  Listeria ivanovii, Listeria seeligeri, Listeria innocua, L. Welshimeri,  or  L. grayi  species. 
     In some embodiments, the  Listeria  are non-naturally occurring. In some embodiments, the  Listeria  are attenuated. In some embodiments, the  Listeria  are viable. In some embodiments, the  Listeria  are mutant  Listeria,  recombinant  Listeria,  or otherwise modified. In some embodiments, the  Listeria  are attenuated. In some embodiments, the  Listeria  are metabolically active. In certain embodiments, the  Listeria  are not infected with bacteriophage. The invention further provides  Listeria  that are recombinant. In addition, the  Listeria  may be isolated and/or substantially purified. 
     In some embodiments, the attenuated  Listeria  is attenuated in one or more of growth, cell to cell spread, binding to or entry into a host cell, replication, or DNA repair. In some embodiments, the  Listeria  is attenuated by one or more of an actA mutation, an inlB mutation, a uvrA mutation, a uvrB mutation, a uvrC mutation, a nucleic acid targeting compound, or a uvrAB mutation and a nucleic acid targeting compound. In some embodiments, the attenuated  Listeria  is attenuated in cell to cell spread and/or entry into nonphagocytic cells. In some embodiments, the  Listeria  is attenuated by one or more of an actA mutation or an actA mutation and an inlB mutation. In some embodiments, the  Listeria  is ΔactA or ΔactAΔinlB. 
     In some embodiments, the attenuated  Listeria  is attenuated for cell-to-cell spread. In some embodiments, the  Listeria  attenuated for cell-to-cell spread are defective with respect to ActA (e.g., relative to the non-modified or wild-type  Listeria ). In some embodiments, the  Listeria  comprises an attenuating mutation in the actA gene. In some embodiments, the  Listeria  comprises a full or partial deletion in the actA gene. 
     In some embodiments, the capacity of the attenuated  Listeria  bacterium for cell-to-cell spread is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%, relative to  Listeria  without the attenuating mutation (e.g., wild type  Listeria ). In some embodiments, the capacity of the attenuated  Listeria  bacterium for cell-to-cell spread is reduced by at least about 25% relative to  Listeria  without the attenuating mutation. In some embodiments, the capacity of the attenuated  Listeria  bacterium attenuated for cell-to-cell spread is reduced by at least about 50% relative to the  Listeria  without the attenuating mutation. 
     In vitro assays for determining whether a  Listeria  bacterium is attenuated for cell-to-cell spread are known to those of ordinary skill in the art. For example, the diameter of plaques formed over a time course after infection of selected cultured cell monolayers can be measured. Plaque assays within L2 cell monolayers can be performed as described previously in Sun, A., A. Camilli, and D. A. Portnoy. 1990, Isolation of  Listeria monocytogenes  small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect. Immun. 58:3770-3778, with modifications to the methods of measurement, as described by in Skoble, J., D. A. Portnoy, and M. D. Welch. 2000, Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and  Listeria monocytogenes  motility.  J Cell Biol.  150:527-538. In brief, L2 cells are grown to confluency in six-well tissue culture dishes and then infected with bacteria for 1 h. Following infection, the cells are overlayed with media warmed to 40° C. that is comprised of DME containing 0.8% agarose, Fetal Bovine Serum (e.g., 2%), and a desired concentration of Gentamicin. The concentration of Gentamicin in the media dramatically affects plaque size, and is a measure of the ability of a selected  Listeria  strain to effect cell-to-cell spread (Glomski, I J., M. M. Gedde, A. W. Tsang, J. A. Swanson, and D. A. Portnoy. 2002. 1 Cell Biol. 156:1029-1038). For example, in some embodiments at 3 days following infection of the monolayer the plaque size of  Listeria  strains having a phenotype of defective cell-to-cell spread is reduced by at least 50% as compared to wild-type  Listeria,  when overlayed with media containing Gentamicin at a concentration of 50 μg/ml. On the other hand, the plaque size between  Listeria  strains having a phenotype of defective cell-to-cell spread and wild-type  Listeria  is similar when infected monolayers are overlayed with media+agarose containing only 5 μg/ml gentamicin. Thus, the relative ability of a selected strain to effect cell-to-cell spread in an infected cell monolayer relative to wild-type  Listeria  can be determined by varying the concentration of gentamicin in the media containing agarose. Optionally, visualization and measurement of plaque diameter can be facilitated by the addition of media containing Neutral Red (GIBCO BRL; 1:250 dilution in DME+agarose media) to the overlay at 48 h. post infection. Additionally, the plaque assay can be performed in monolayers derived from other primary cells or continuous cells. For example HepG2 cells, a hepatocyte-derived cell line, or primary human hepatocytes can be used to evaluate the ability of selected  Listeria  mutants to effect cell-to-cell spread, as compared to wild-type  Listeria.  In some embodiments,  Listeria  comprising mutations or other modifications that attenuate the  Listeria  for cell-to-cell spread produce “pinpoint” plaques at high concentrations of gentamicin (about 50 μg/ml). 
     In some embodiments, the  Listeria  is attenuated for entry into non-phagocytic cells (relative or the non-mutant or wildtype  Listeria ). In some embodiments, the  Listeria  is defective with respect to one or more internalins (or equivalents). In some embodiments, the  Listeria  is defective with respect to internalin A. In some embodiments, the  Listeria  is defective with respect to internalin B. In some embodiments, the  Listeria  comprise a mutation in inlA. In some embodiments, the  Listeria  comprise a mutation in inlB. In some embodiments, the  Listeria  comprise a mutation in both actA and inlB. In some embodiments, the  Listeria  is deleted in functional ActA and internalinB. In some embodiments, the attenuated  Listeria  bacterium is an ΔactAΔinlB double deletion mutant. In some embodiments, the  Listeria  bacterium is defective with respect to both ActA and internalin B. 
     In some embodiments, the capacity of the attenuated  Listeria  bacterium for entry into non-phagocytic cells is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%, relative to  Listeria  without the attenuating mutation (e.g., the wild type bacterium). In some embodiments, the capacity of the attenuated  Listeria  bacterium for entry into non-phagocytic cells is reduced by at least about 25% relative to  Listeria  without the attenuating mutation. In some embodiments, the capacity of the attenuated bacterium for entry into non-phagocytic cells is reduced by at least about 50% relative to  Listeria  without the attenuating mutation. In some embodiments, the capacity of the attenuated  Listeria  bacterium for entry into non-phagocytic cells is reduced by at least about 75% relative to  Listeria  without the attenuating mutation. 
     In some embodiments, the attenuated  Listeria  is not attenuated for entry into more than one type of non-phagocytic cell. For instance, the attenuated strain may be attenuated for entry into hepatocytes, but not attenuated for entry into epithelial cells. As another example, the attenuated strain may be attenuated for entry into epithelial cells, but not hepatocytes. It is also understood that attenuation for entry into a non-phagocytic cell of a particular modified  Listeria  is a result of mutating a designated gene, for example a deletion mutation, encoding an invasin protein which interacts with a particular cellular receptor, and as a result facilitates infection of a non-phagocytic cell. For example,  Listeria  ΔinlB mutant strains are attenuated for entry into non-phagocytic cells expressing the hepatocyte growth factor receptor (c-met), including hepatocyte cell lines (e.g., HepG2), and primary human hepatocytes. 
     In some embodiments, even though the  Listeria  is attenuated for entry into non-phagocytic cells, the  Listeria  is still capable of uptake by phagocytic cells, such as at least dendritic cells and/or macrophages. In one embodiment the ability of the attenuated  Listeria  to enter phagocytic cells is not diminished by the modification made to the strain, such as the mutation of an invasin (i.e. approximately 95% or more of the measured ability of the strain to be taken up by phagocytic cells is maintained post-modification). In other embodiments, the ability of the attenuated  Listeria  to enter phagocytic cells is diminished by no more than about 10%, no more than about 25%, no more than about 50%, or no more than about 75%. 
     In some embodiments of the invention, the amount of attenuation in the ability of the  Listeria  to enter non-phagocytic cells ranges from a two-fold reduction to much greater levels of attenuation. In some embodiments, the attenuation in the ability of the  Listeria  to enter non-phagocytic cells is at least about 0.3 log, about 1 log, about 2 log, about 3 log, about 4 log, about 5 log, or at least about 6 log. In some embodiments, the attenuation is in the range of about 0.3 to &gt;8 log, about 2 to &gt;8 log, about 4 to &gt;8 log, about 6 to &gt;8 log, about 0.3-8 log, also about 0.3-7 log, also about 0.3-6 log, also about 0.3-5 log, also about 0.3-4 log, also about 0.3-3 log, also about 0.3-2 log, also about 0.3-1 log. In some embodiments, the attenuation is in the range of about 1 to &gt;8 log, 1-7 log, 1-6 log, also about 2-6 log, also about 2-5 log, also about 3-5 log. 
     In vitro assays for determining whether or not a  Listeria  bacterium is attenuated for entry into non-phagocytic cells are known to those of ordinary skill in the art. For instance, both Dramsi et al.,  Molecular Microbiology  16:251-261 (1995) and Gaillard et al.,  Cell  65:1127-1141 (1991) describe assays for screening the ability of mutant  L. monocytogenes  strains to enter certain cell lines. For instance, to determine whether a  Listeria  bacterium with a particular modification is attenuated for entry into a particular type of non-phagocytic cells, the ability of the attenuated  Listeria  bacterium to enter a particular type of non-phagocytic cell is determined and compared to the ability of the identical  Listeria  bacterium without the modification to enter non-phagocytic cells. Likewise, to determine whether a  Listeria  strain with a particular mutation is attenuated for entry into a particular type of non-phagocytic cells, the ability of the mutant  Listeria  strain to enter a particular type of non-phagocytic cell is determined and compared to the ability of the  Listeria  strain without the mutation to enter non-phagocytic cells. For instance, the ability of a modified  Listeria  bacterium to infect non-phagocytic cells, such as hepatocytes, can be compared to the ability of non-modified  Listeria  or wild type  Listeria  to infect phagocytic cells. In such an assay, the modified and non-modified  Listeria  is typically added to the non-phagocytic cells in vitro for a limited period of time (for instance, an hour), the cells are then washed with a gentamicin-containing solution to kill any extracellular bacteria, the cells are lysed and then plated to assess titer. Examples of such an assay are found in U.S. Patent Publication No. 2004/0228877. In addition, confirmation that the strain is defective with respect to internalin B may also be obtained through comparison of the phenotype of the strain with the previously reported phenotypes for internalin B mutants. 
     A  Listeria monocytogenes  ΔactAΔinlB strain was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, United States of America (P.O. Box 1549, Manassas, Va., 20108, United States of America), on Oct. 3, 2003, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and designated with accession number PTA-5562. Another  Listeria monocytogenes  strain, an ΔactA ΔuvrAB strain, was also deposited with the ATCC on Oct. 3, 2003, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and designated with accession number PTA-5563. 
     In some embodiments,  Listeria  is attenuated for nucleic acid repair (e.g., relative to wildtype). For instance, in some embodiments, the  Listeria  is defective with respect to at least one DNA repair enzyme (e.g.,  Listeria monocytogenes  uvrAB mutants). In some embodiments, the  Listeria  is defective with respect to PhrB, UvrA, UvrB, UvrC, UvrD, and/or RecA. In some embodiments, the bacteria are defective with respect to UvrA, UvrB, and/or UvrC. In some embodiments, the bacteria comprise attenuating mutations in phrB, uvrA, uvrB, uvrC, uvrD, and/or recA genes. In some embodiments, the bacteria comprise one or more mutations in the uvrA, uvrB, and/or uvrC genes. In some embodiments, the bacteria are functionally deleted in UvrA, UvrB, and/or UvrC. In some embodiments, the bacteria are deleted in functional UvrA and UvrB. In some embodiments, the bacteria are uvrAB deletion mutants. In some embodiments, the bacteria are ΔuvrABΔactA mutants. In some embodiments, the nucleic acid of the bacteia which are attenuated for nucleic acid repair and/or are defective with respect to at least one DNA repair enzyme are modified by reaction with a nucleic acid targeting compound. Nucleic acid repair mutants, such as ΔuvrAB  Listeria monocytogenes  mutants, and methods of making the mutants, are described in detail in U.S. Patent Publication No. 2004/0197343, which is incorporated by reference herein in its entirety (see, e.g., Example 7 of U.S. 2004/0197343). 
     In some embodiments, the capacity of the attenuated  Listeria  bacterium for nucleic acid repair is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%, relative to a  Listeria  bacterium without the attenuating mutation (e.g., the wild type bacterium). In some embodiments, the capacity of the attenuated  Listeria  bacterium for nucleic acid repair is reduced by at least about 25% relative to a  Listeria  bacterium without the attenuating mutation. In some embodiments, the capacity of the attenuated  Listeria  bacterium attenuated for nucleic acid repair is reduced by at least about 50% relative a  Listeria  bacterium without the attenuating mutation. 
     Confirmation that a particular mutation is present in a bacterial strain can be obtained through a variety of methods known to those of ordinary skill in the art. For instance, the relevant portion of the strain&#39;s genome can be cloned and sequenced. Alternatively, specific mutations can be identified via PCR using paired primers that code for regions adjacent to a deletion or other mutation. Southern blots can also be used to detect changes in the bacterial genome. Also, one can analyze whether a particular protein is expressed by the strain using techniques standard to the art such as Western blotting. Confirmation that the strain contains a mutation in the desired gene may also be obtained through comparison of the phenotype of the strain with a previously reported phenotype. For example, the presence of a nucleotide excision repair mutation such as deletion of uvrAB can be assessed using an assay which tests the ability of the bacteria to repair its nucleic acid using the nucleotide excision repair (NER) machinery and comparing that ability against wild-type bacteria. Such functional assays are known in the art. For instance, cyclobutane dimer excision or the excision of UV-induced (6-4) products can be measured to determine a deficiency in an NER enzyme in the mutant (see, e.g., Franklin et al.,  Proc. Natl. Acad. Sci. USA,  81: 3821-3824 (1984)). Alternatively, survival measurements can be made to assess a deficiency in nucleic acid repair. For instance, the  Listeria  can be subjected to psoralen/UVA treatment and then assessed for their ability to proliferate and/or survive in comparison to wild-type. 
     The invention supplies a number of  Listeria  strains for making or engineering an attenuated  Listeria  of the present invention (Table 8). The  Listeria  of the present invention are not to be limited by the strains disclosed in this table. 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Exemplary strains of  Listeria  for use as parental 
               
               
                 strains in the present invention. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   L. monocytogenes  10403S wild type. 
                 Bishop and Hinrichs (1987) J. Immunol. 
               
               
                   
                 139: 2005-2009; Lauer, et al. (2002) J. 
               
               
                   
                 Bact. 184: 4177-4186. 
               
               
                   L. monocytogenes  DP-L4056 (phage cured). 
                 Lauer, et al. (2002) J. Bact. 184: 4177-4186. 
               
               
                 The prophage-cured 10403S strain is 
               
               
                 designated DP-L4056. 
               
               
                   L. monocytogenes  DP-L4027, which is 
                 Lauer, et al. (2002) J. Bact. 184: 4177-4186; 
               
               
                 DP-L2161, phage cured, deleted in hly gene. 
                 Jones and Portnoy (1994) Infect. 
               
               
                   
                 Immunity 65: 5608-5613. 
               
               
                   L. monocytogenes  DP-L4029, which is DP- 
                 Lauer, et al. (2002) J. Bact. 184: 4177-4186; 
               
               
                 L3078, phage cured, deleted in actA. 
                 Skoble, et al. (2000) J. Cell Biol. 
               
               
                   
                 150: 527-538. 
               
               
                   L. monocytogenes  DP-L4042 (delta PEST) 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                   
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  DP-L4097 (LLO-S44A). 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                   
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  DP-L4364 (delta lplA; 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                 lipoate protein ligase). 
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  DP-L4405 (delta inlA). 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                   
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  DP-L4406 (delta inlB). 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                   
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  CS-L0001 (delta actA- 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                 delta inlB). 
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  CS-L0002 (delta actA- 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                 delta lplA). 
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  CS-L0003 (L461T-delta 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                 lplA). 
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  DP-L4038 (delta actA- 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                 LLO L461T). 
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes  DP-L4384 (S44A-LLO 
                 Brockstedt, et al. (2004) Proc. Natl. Acad. 
               
               
                 L461T). 
                 Sci. USA 101: 13832-13837; supporting 
               
               
                   
                 information. 
               
               
                   L. monocytogenes . Mutation in lipoate 
                 O&#39;Riordan, et al. (2003) Science 302: 462-464. 
               
               
                 protein ligase (LplA1). 
               
               
                   L. monocytogenes  DP-L4017 (10403S with 
                 U.S. Provisional Pat. Appl. Ser. No. 
               
               
                 LLO L461T point mutation in hemolysin 
                 60/490,089 filed Jul. 24, 2003. 
               
               
                 gene). 
               
               
                   L. monocytogenes  EGD. 
                 GenBank Acc. No. AL591824. 
               
               
                   L. monocytogenes  EGD-e. 
                 GenBank Acc. No. NC_003210. ATCC 
               
               
                   
                 Acc. No. BAA-679. 
               
               
                   L. monocytogenes  strain EGD, complete 
                 GenBank Acc. No. AL591975 
               
               
                 genome, segment 3/12 
               
               
                   L. monocytogenes . 
                 ATCC Nos. 13932; 15313; 19111-19120; 
               
               
                   
                 43248-43251; 51772-51782. 
               
               
                   L. monocytogenes  DP-L4029 deleted 
                 U.S. Provisional Pat. Appl. Ser. No. 
               
               
                 in uvrAB. 
                 60/541,515 filed Feb. 2, 2004; U.S. 
               
               
                   
                 Provisional Pat. Appl. Ser. No. 60/490,080 
               
               
                   
                 filed Jul. 24, 2003. 
               
               
                   L. monocytogenes  DP-L4029 deleted 
                 U.S. Provisional Pat. Appl. Ser. No. 
               
               
                 in uvrAB treated with a psoralen. 
                 60/541,515 filed Feb. 2, 2004. 
               
               
                   L. monocytogenes  actA − /inlB −  double mutant. 
                 Deposited with ATCC on Oct. 3, 2003. 
               
               
                   
                 Acc. No. PTA-5562. 
               
               
                   L. monocytogenes  lplA mutant or hly 
                 U.S. Pat. Applic. No. 20040013690 of 
               
               
                 mutant. 
                 Portnoy, et al. 
               
               
                   L. monocytogenes  DAL/DAT double 
                 U.S. Pat. Applic. No. 20050048081 of 
               
               
                 mutant. 
                 Frankel and Portnoy. 
               
               
                   L. monocytogenes  str. 4b F2365. 
                 GenBank Acc. No. NC_002973. 
               
               
                 
                   Listeria ivanovii 
                 
                 ATCC No. 49954 
               
               
                   Listeria innocua  Clip11262. 
                 GenBank Acc. No. NC_003212; 
               
               
                   
                 AL592022. 
               
               
                   Listeria innocua , a naturally occurring 
                 Johnson, et al. (2004) Appl. Environ. 
               
               
                 hemolytic strain containing the 
                 Microbiol. 70: 4256-4266. 
               
               
                 PrfA-regulated virulence gene cluster. 
               
               
                   Listeria seeligeri . 
                 Howard, et al. (1992) Appl. Eviron. 
               
               
                   
                 Microbiol. 58: 709-712. 
               
               
                   Listeria innocua  with  L. monocytogenes   
                 Johnson, et al. (2004) Appl. Environ. 
               
               
                 pathogenicity island genes. 
                 Microbiol. 70: 4256-4266. 
               
               
                   Listeria innocua  with  L. monocytogenes   
                 See, e.g., Lingnau, et al. (1995) Infection 
               
               
                 internalin A gene, e.g., as a plasmid or as a 
                 Immunity 63: 3896-3903; Gaillard, et al. 
               
               
                 genomic nucleic acid. 
                 (1991) Cell 65: 1127-1141). 
               
               
                   
               
            
           
         
       
     
     The present invention encompasses reagents and methods that comprise the above listerial strains, as well as these strains that are modified, e.g., by a plasmid and/or by genomic integration, to contain a nucleic acid encoding one of, or any combination of, the following genes: hly (LLO; listeriolysin); iap (p60); inlA; inlB; inlC; dal (alanine racemase); daaA (dat; D-amino acid aminotransferase); plcA; plcB; actA; or any nucleic acid that mediates growth, spread, breakdown of a single walled vesicle, breakdown of a double walled vesicle, binding to a host cell, uptake by a host cell. The present invention is not to be limited by the particular strains disclosed above. 
     In some embodiments, the attenuation of  Listeria  can be measured in terms of biological effects of the  Listeria  on a host. The pathogenicity of a strain can be assessed by measurement of the LD 50  in mice or other vertebrates. The LD 50  is the amount, or dosage, of  Listeria  injected into vertebrates necessary to cause death in 50% of the vertebrates. The LD 50  values can be compared for bacteria having a particular modification (e.g., mutation) versus the bacteria without the particular modification as a measure of the level of attenuation. For example, if the bacterial strain without a particular mutation has an LD 50  of 10 3  bacteria and the bacterial strain having the particular mutation has an LD 50  of 10 5  bacteria, the strain has been attenuated so that is LD 50  is increased 100-fold or by 2 log. 
     In some embodiments, the attenuated  Listeria  has an LD 50  that is at least about 5 times higher, at least about 10 times higher, at least about 100 times higher, at least about 1000 times higher, or at least about 1×10 4  higher than the LD 50  of parental or wildtype  Listeria.    
     As a further example, the degree of attenuation may also be measured qualitatively by other biological effects, such as the extent of tissue pathology or serum liver enzyme levels. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin and bilirubin levels in the serum are determined at a clinical laboratory for mice injected with  Listeria  (or other bacteria). Comparisons of these effects in mice or other vertebrates can be made for  Listeria  with and without particular modifications/mutations as a way to assess the attenuation of the  Listeria.  Attenuation of the  Listeria  may also be measured by tissue pathology. The amount of  Listeria  that can be recovered from various tissues of an infected vertebrate, such as the liver, spleen and nervous system, can also be used as a measure of the level of attenuation by comparing these values in vertebrates injected with mutant versus non-mutant  Listeria.  For instance, the amount of  Listeria  that can be recovered from infected tissues such as liver or spleen as a function of time can be used as a measure of attenuation by comparing these values in mice injected with mutant vs. non-mutant  Listeria.    
     Accordingly, the attenuation of the  Listeria  can be measured in terms of bacterial load in particular selected organs in mice known to be targets by wild-type  Listeria.  For example, the attenuation of the  Listeria  can be measured by enumerating the colonies (Colony Forming Units; CFU or cfu) arising from plating dilutions of liver or spleen homogenates (homogenized in H 2 0+0.2% NP40) on BHI agar media. The liver or spleen cfu can be measured, for example, over a time course following administration of the modified  Listeria  via any number of routes, including intravenous, intraperitoneal, intramuscular, and subcutaneous. Additionally, the  Listeria  can be measured and compared to a drug-resistant, wild type  Listeria  (or any other selected  Listeria  strain) in the liver and spleen (or any other selected organ) over a time course following administration by the competitive index assay, as described. 
     Methods of producing mutant  Listeria  are well known in the art. Bacterial mutations can be achieved through traditional mutagenic methods, such as mutagenic chemicals or radiation followed by selection of mutants. Bacterial mutations can also be achieved by one of skill in the art through recombinant DNA technology. For instance, the method of allelic exchange using the pKSV7 vector described in Camilli et al.,  Molecular Micro.  8:143-157 (1993) is suitable for use in generating mutants including deletion mutants. (Camilli et al. (1993) is incorporated by reference herein in its entirety.) Alternatively, the gene replacement protocol described in Biswas et al.,  J. Bacteriol.  175:3628-3635 (1993), can be used. Other similar methods are known to those of ordinary skill in the art. 
     A variety of bacterial mutants, and their construction, are described in U.S. patent application Ser. No. 10/883,599, U.S. Patent Publication No. 2004/0197343, U.S. Patent Publication No. 2005/0249748, and U.S. Patent Publication No. 2004/0228877, each of which is incorporated by reference herein in its entirety. 
     The degree of attenuation in uptake of the attenuated bacteria by non-phagocytic cells need not be an absolute attenuation in order to provide a safe and effective vaccine. In some embodiments, the degree of attenuation is one that provides for a reduction in toxicity sufficient to prevent or reduce the symptoms of toxicity to levels that are not life threatening. 
     In some embodiments, the  Listeria  cannot form colonies, replicate, and/or divide. In some embodiments of the invention, the  Listeria  is attenuated for proliferation relative to parental or wildtype  Listeria.    
     In some embodiments, the attenuated  Listeria  is killed, but metabolically active (US Patent Pub. No. 2004/0197343 and Brockstedt, et al., Nat. Med., 11:853-60 (2005), incorporated by reference herein in its entirety). 
     The  Listeria,  may, in some embodiments, be attenuated by a nucleic acid targeting compound. In some embodiments, the nucleic-acid targeting compound is a nucleic acid alkylator, such as β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. In some embodiments, the nucleic acid targeting compound is activated by irradiation, such as UVA irradiation. In some embodiments, the  Listeria  is treated with a psoralen compound. For instance, in some embodiments, the bacterium are modified by treatment with a psoralen, such as 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen (“S-59”), and UVA light. In some embodiments, the nucleic acid of the bacterium has been modified by treatment with a psoralen compound and UVA irradiation. Descriptions of methods of modifying bacteria to attenuate them for proliferation using nucleic acid targeting compounds are described in U.S. Patent Pub. No. 2004/0197343 and Brockstedt, et al., Nat. Med., 11:853-60 (2005). In some embodiments, the  Listeria  is attenuated for DNA repair. 
     For example, for treatment of  Listeria  such as ΔactAΔuvrAB  L. monocytogenes,  in some embodiments, S-59 psoralen can be added to 200 nM in a log-phase culture of (approximately) OD 600 =0.5, followed by inactivation with 6 J/m 2  of UVA light when the culture reaches an optical density of one. Inactivation conditions are optimized by varying concentrations of S-59, UVA dose, the time of S-59 exposure prior to UVA treatment as well as varying the time of treatment during bacterial growth of the  Listeria  actA/uvrAB strain. The parental  Listeria  strain is used as a control. Inactivation of  Listeria  (log-kill) is determined by the inability of the bacteria to form colonies on BHI (Brain heart infusion) agar plates. In addition, one can confirm the continued metabolic activity and expression of proteins such as LLO in the bacteria in the S-59/UVA inactivated  Listeria  using  35 5-pulse-chase experiments to determine the synthesis and secretion of newly expressed proteins post S-59/UVA inactivation. Expression of LLO using  35 S-metabolic labeling can be routinely determined. S-59/UVA inactivated  Listeria  actA/uvrAB can be incubated for 1 hour in the presence of  35 S-Methionine. Expression and/or secretion of proteins such as LLO can be determined of both whole cell lysates, and TCA precipitation of bacterial culture fluids. LLO-specific monoclonal antibodies can be used for immunoprecipitation to verify the continued expression and secretion from recombinant  Listeria  post inactivation. 
     In some embodiments, the  Listeria  attenuated for proliferation are also attenuated for nucleic acid repair and/or are defective with respect to at least one DNA repair enzyme. For instance, in some embodiments, the bacterium in which nucleic acid has been modified by a nucleic acid targeting compound such as a psoralen (combined with UVA treatment) is a uvrAB deletion mutant. 
     In some embodiments, the proliferation of the  Listeria  is attenuated by at least about 0.3 log, also at least about 1 log, about 2 log, about 3 log, about 4 log, about 6 log, or at least about 8 log. In another embodiment, the proliferation of the  Listeria  is attenuated by about 0.3 to &gt;10 log, about 2 to &gt;10 log, about 4 to &gt;10 log, about 6 to &gt;10 log, about 0.3-8 log, about 0.3-6 log, about 0.3-5 log, about 1-5 log, or about 2-5 log. In some embodiments, the expression of LLO by the  Listeria  is at least about 10%, about 25%, about 50%, about 75%, or at least about 90% of the expression of LLO in non-modified  Listeria.    
     V. Pharmaceutical Compositions, Immunogenic Compositions, and/or Vaccines 
     A variety of different compositions such as pharmaceutical compositions, immunogenic compositions, and vaccines comprising the  Listeria  described herein are also provided by the invention. In some embodiments, the compositions are isolated. 
     As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Pharmaceutically acceptable carriers are well known to those of ordinary skill in the art, and include any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject&#39;s immune system. For instance, pharmaceutically acceptable carriers include, but are not limited to, water, buffered saline solutions (e.g., 0.9% saline), emulsions such as oil/water emulsions, and various types of wetting agents. Possible carriers also include, but are not limited to, oils (e.g., mineral oil), dextrose solutions, glycerol solutions, chalk, starch, salts, glycerol, and gelatin. 
     While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. In some embodiments, for parenteral administration, such as subcutaneous injection, the carrier comprises water, saline, alcohol, a fat, a wax or a buffer. In some embodiments, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, are employed for oral administration. 
     Compositions comprising such carriers are formulated by well known conventional methods (see, for example,  Remington&#39;s Pharmaceutical Sciences,  18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and  Remington, The Science and Practice of Pharmacy  20th Ed. Mack Publishing, 2000). 
     In addition to pharmaceutical compositions, immunogenic compositions are provided. For instance, the invention provides an immunogenic composition comprising a recombinant bacterium described herein. 
     In some embodiments, the recombinant bacterium in the immunogenic composition releases the polypeptide comprising the antigen at a level sufficient to induce an immune response to the antigen upon administration of the composition to a host (e.g., a mammal such as a human). In some embodiments, the immune response stimulated by the immunogenic composition is a cell-mediated immune response. In some embodiments, the immune response stimulated by the immunogenic composition is a humoral immune response. In some embodiments, the immune response stimulated by the immunogenic composition comprises both a humoral and cell-mediated immune response. 
     It can be determined if a particular form of recombinant bacteria (and/or a particular expression cassette) is useful in an immunogenic composition (or as a vaccine) by testing the ability of the recombinant bacteria to stimulate an immune response in vitro or in a model system. 
     These immune cell responses can be measured by both in vitro and in vivo methods to determine if the immune response of a particular recombinant bacterium (and/or a particular expression cassette) is effective. One possibility is to measure the presentation of the protein or antigen of interest by an antigen-presenting cell that has been mixed with a population of the recombinant bacteria. The recombinant bacteria may be mixed with a suitable antigen presenting cell or cell line, for example a dendritic cell, and the antigen presentation by the dendritic cell to a T cell that recognizes the protein or antigen can be measured. If the recombinant bacteria are expressing the protein or antigen at a sufficient level, it will be processed into peptide fragments by the dendritic cells and presented in the context of MHC class I or class II to T cells. For the purpose of detecting the presented protein or antigen, a T cell clone or T cell line responsive to the particular protein or antigen may be used. The T cell may also be a T cell hybridoma, where the T cell is immortalized by fusion with a cancer cell line. Such T cell hybridomas, T cell clones, or T cell lines can comprise either CD8+ or CD4+ T cells. The dendritic cell can present to either CD8+ or CD4+ T cells, depending on the pathway by which the antigens are processed. CD8+ T cells recognize antigens in the context of MHC class I while CD4+ recognize antigens in the context of MHC class II. The T cell will be stimulated by the presented antigen through specific recognition by its T cell receptor, resulting in the production of certain proteins, such as IL-2, tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ), that can be quantitatively measured (for example, using an ELISA assay, ELISPOT assay, or Intracellular Cytokine Staining (ICS)). These are techniques that are well known in the art. 
     Alternatively, a hybridoma can be designed to include a reporter gene, such as β-galactosidase, that is activated upon stimulation of the T cell hybridoma by the presented antigens. The increase in the production of β-galactosidase can be readily measured by its activity on a substrate, such as chlorophenol red-B-galactoside, which results in a color change. The color change can be directly measured as an indicator of specific antigen presentation. 
     Additional in vitro and in vivo methods for assessing the antigen expression of recombinant bacteria vaccines of the present invention are known to those of ordinary skill in the art. It is also possible to directly measure the expression of a particular heterologous antigen by recombinant bacteria. For example, a radioactively labeled amino acid can be added to a cell population and the amount of radioactivity incorporated into a particular protein can be determined. The proteins synthesized by the cell population can be isolated, for example by gel electrophoresis or capillary electrophoresis, and the amount of radioactivity can be quantitatively measured to assess the expression level of the particular protein. Alternatively, the proteins can be expressed without radioactivity and visualized by various methods, such as an ELISA assay or by gel electrophoresis and Western blot with detection using an enzyme linked antibody or fluorescently labeled antibody. 
     Elispot assay, Intracellular Cytokine Staining Assay (ICS), measurement of cytokine expression of stimulated spleen cells, and assessment of cytotoxic T cell activity in vitro and in vivo are all techniques for assessing immunogenicity known to those in the art. 
     In addition, therapeutic efficacy of the vaccine composition can be assessed more directly by administration of the immunogenic composition or vaccine to an animal model such as a mouse model, followed by an assessment of survival or tumor growth. For instance, survival can be measured following administration of the  Listeria  and challenge. 
     Mouse models useful for testing the immunogenicity of an immunogenic composition or vaccine expressing a particular antigen can be produced by first modifying a tumor cell so that it expresses the antigen of interest or a model antigen and then implanting the tumor cells expressing the antigen of interest into mice. The mice can be vaccinated with the candidate immunogenic composition or vaccine comprising a recombinant bacterium expressing a polypeptide comprising the antigen of interest or a model antigen prior to implantation of the tumor cells (to test prophylactic efficacy of the candidate composition) or following implantation of the tumor cells in the mice (to test therapeutic efficacy of the candidate composition). 
     As an example, CT26 mouse murine colon carcinoma cells can be transfected with an appropriate vector comprising an expression cassette encoding the desired antigen or model antigen using techniques standard in the art. Standard techniques such as flow cytometry and Western blots can then be used to identify clones expressing the antigen or model antigen at sufficient levels for use in the immunogenicity and/or efficacy assays. 
     Alternatively, candidate compositions can be tested which comprise a recombinant bacterium expressing an antigen that corresponds to or is derived from an antigen endogenous to a tumor cell line (e.g., the retroviral gp70 tumor antigen AH1 endogenous to CT26 mouse murine colon carcinoma cells, or the heteroclitic epitope AH1-A5). In such assays, the tumor cells can be implanted in the animal model without further modification to express an additional antigen. Candidate vaccines comprising the antigen can then be tested. 
     As indicated, vaccine compositions comprising the bacteria described herein are also provided. 
     In some embodiments, the vaccine compositions comprise antigen-presenting cells (APC) which have been infected with any of the recombinant bacteria described herein. In some embodiments the vaccine (or immunogenic or pharmaceutical composition) does not comprise antigen-presenting cells (i.e., the vaccine or composition is a bacteria-based vaccine or composition, not an APC-based vaccine or composition). 
     Methods of administration suitable for administration of vaccine compositions (and pharmaceutical and immunogenic compositions) are known in the art, and include oral, intraveneous, intradermal, intraperitoneal, intramuscular, intralymphatic, intranasal and subcutaneous routes of administration. 
     Vaccine formulations are known in the art and in some embodiments may include numerous additives, such as preservatives (e.g., thimerosal, 2-phenyoyx ethanol), stabilizers, adjuvants (e.g. aluminum hydroxide, aluminum phosphate, cytokines), antibiotics (e.g., neomycin, streptomycin), and other substances. In some embodiments, stabilizers, such as lactose or monosodium glutamate (MSG), are added to stabilize the vaccine formulation against a variety of conditions, such as temperature variations or a freeze-drying process. In some embodiments, vaccine formulations may also include a suspending fluid or diluent such as sterile water, saline, or isotonic buffered saline (e.g., phosphate buffered to physiological pH). Vaccine may also contain small amount of residual materials from the manufacturing process. 
     For instance, in some embodiments, the vaccine compositions are lyophilized (i.e., freeze-dried). The lyophilized preparation can be combined with a sterile solution (e.g., citrate-bicarbonate buffer, buffered water, 0.4% saline, or the like) prior to administration. 
     In some embodiments, the vaccine compositions may further comprise additional components known in the art to improve the immune response to a vaccine, such as adjuvants or co-stimulatory molecules. In addition to those listed above, possible adjuvants include chemokines and bacterial nucleic acid sequences, like CpG. In some embodiments, the vaccines comprise antibodies that improve the immune response to a vaccine, such as CTLA4. In some embodiments, co-stimulatory molecules comprise one or more factors selected from the group consisting of GM-CSF, IL-2, IL-12, IL-14, IL-15, IL-18, B7.1, B7.2, and B7-DC are optionally included in the vaccine compositions of the present invention. Other co-stimulatory molecules are known to those of ordinary skill in the art. 
     In additional aspects, the invention provides methods of improving a vaccine or immunogenic composition comprising  Listeria  that express an antigen. 
     Methods of producing the vaccines of the present invention are also provided. 
     VI. Uses 
     A variety of methods of using the  Listeria  or pharmaceutical, immunogenic, or vaccine compositions described herein for inducing immune responses, and/or preventing or treating conditions in a host (e.g., a mammal) are provided. In some embodiments, the condition that is treated or prevented is a disease. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease. 
     As used herein, “treatment” or “treating” (with respect to a condition or a disease) encompasses an approach for obtaining beneficial or desired results. In some embodiments, these results include clinical results. For purposes of this invention, beneficial or desired results with respect to a disease may include, but are not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, and/or prolonging survival. Likewise, for purposes of this invention, beneficial or desired results with respect to a condition may include, but are not limited to, one or more of the following: improving a condition, curing a condition, lessening severity of a condition, delaying progression of a condition, alleviating one or more symptoms associated with a condition, increasing the quality of life of one suffering from a condition, and/or prolonging survival. For instance, in those embodiments where the compositions described herein are used for treatment of cancer, the beneficial or desired results may include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, reducing metastasis of neoplastic cells found in cancers, shrinking the size of a tumor, decreasing symptoms resulting from the cancer, increasing the quality of life of those suffering from the cancer, decreasing the dose of other medications required to treat the disease, delaying the progression of the cancer, and/or prolonging survival of patients having cancer. 
     As used herein, the terms “preventing” disease or “protecting a host” from disease (used interchangeably herein) encompass, but are not limited to, one or more of the following: stopping, deferring, hindering, slowing, retarding, and/or postponing the onset or progression of a disease, stabilizing the progression of a disease, and/or delaying development of a disease. The terms “preventing” a condition or “protecting a host” from a condition (used interchangeably herein) encompass, but are not limited to, one or more of the following: stopping, deferring, hindering, slowing, retarding, and/or postponing the onset or progression of a condition, stabilizing the progression of a condition, and/or delaying development of a condition. The period of this prevention can be of varying lengths of time, depending on the history of the disease or condition and/or individual being treated. By way of example, where the vaccine is designed to prevent or protect against an infectious disease caused by a pathogen, the terms “preventing” disease or “protecting a host” from disease encompass, but are not limited to, one or more of the following: stopping, deferring, hindering, slowing, retarding, and/or postponing the infection by a pathogen of a host, progression of an infection by a pathogen of a host, or the onset or progression of a disease associated with infection of a host by a pathogen, and/or stabilizing the progression of a disease associated with infection of a host by a pathogen. Also, by way of example, where the vaccine is an anti-cancer vaccine, the terms “preventing” disease or.“protecting the host” from disease encompass, but are not limited to, one or more of the following: stopping, deferring, hindering, slowing, retarding, and/or postponing the development of cancer or metastasis, progression of a cancer, or a reoccurrence of a cancer. 
     In one aspect, the invention provides a method of inducing an immune response in a host (e.g., mammal) to an antigen, comprising administering to the host an effective amount of a bacterium described herein or an effective amount of a composition (e.g., a pharmaceutical composition, immunogenic composition, or vaccine) comprising a bacterium described herein. 
     In some embodiments, the immune response is an MHC Class I immune response. In other embodiments, the immune response is an MHC Class II immune response. In still other embodiments, the immune response that is induced by administration of the bacteria or compositions is both an MHC Class I and an MHC Class II response. Accordingly, in some embodiments, the immune response comprises a CD4+ T-cell response. In some embodiments, the immune response comprises a CD8+ T-cell response. In some embodiments, the immune response comprises both a CD4+ T-cell response and a CD8+ T-cell response. In some embodiments, the immune response comprises a B-cell response and/or a T-cell response. B-cell responses may be measured by determining the titer of an antibody directed against the antigen, using methods known to those of ordinary skill in the art. In some embodiments, the immune response which is induced by the compositions described herein is a humoral response. In other embodiments, the immune response which is induced is a cellular immune response. In some embodiments, the immune response comprises both cellular and humoral immune responses. In some embodiments, the immune response is antigen-specific. In some embodiments, the immune response is an antigen-specific T-cell response. 
     In addition to providing methods of inducing immune responses, the present invention also provides methods of preventing or treating a condition or disease in a host (e.g., a mammalian subject such as human patient). The methods comprise administration to the host of an effective amount of a bacterium described herein, or a composition comprising a bacterium described herein. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease. 
     In some embodiments, the disease is cancer. In some embodiments, where the condition being treated or prevented is cancer, the disease is melanoma, breast cancer, pancreatic cancer, liver cancer, colon cancer, colorectal cancer, lung cancer, brain cancer, testicular cancer, ovarian cancer, squamous cell cancer, gastrointestinal cancer, cervical cancer, kidney cancer, thyroid cancer or prostate cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is metastatic. 
     In other embodiments, the disease is an infectious disease or another disease caused by a pathogen such as a virus, bacterium, fungus, or protozoa. In some embodiments, the disease is an infectious disease. 
     In some embodiments, the use of the  Listeria  in the prophylaxis or treatment of a cancer comprises the delivery of the  Listeria  to cells of the immune system of an individual to prevent or treat a cancer present or to which the individual has increased risk factors, such as environmental exposure and/or familial disposition. In other embodiments, the use of the bacteria in the prophylaxis or treatment of a cancer comprises delivery of the bacteria to an individual who has had a tumor removed or has had cancer in the past, but is currently in remission. 
     In some embodiments, administration of composition comprising a bacterium described herein to a host elicits a CD4+ T-cell response in the host. In some other embodiments, administration of a composition comprising a bacterium described herein to a host elicits a CD8+ T-cell response in the host. In some embodiments, administration of a composition comprising a bacterium described herein elicits both a CD4+ T-cell response and a CD8+ T-cell response in the host. 
     The efficacy of the vaccines or other compositions for the treatment of a condition can be evaluated in an individual, for example in mice. A mouse model is recognized as a model for efficacy in humans and is useful in assessing and defining the vaccines of the present invention. The mouse model is used to demonstrate the potential for the effectiveness of the vaccines in any individual. Vaccines can be evaluated for their ability to provide either a prophylactic or therapeutic effect against a particular disease. For example, in the case of infectious diseases, a population of mice can be vaccinated with a desired amount of the appropriate vaccine of the invention, where the bacterium expresses an infectious disease associated antigen. The mice can be subsequently infected with the infectious agent related to the vaccine antigen and assessed for protection against infection. The progression of the infectious disease can be observed relative to a control population (either non vaccinated or vaccinated with vehicle only or a bacterium that does not contain the appropriate antigen). 
     In the case of cancer vaccines, tumor cell models are available, where a tumor cell line expressing a desired tumor antigen can be injected into a population of mice either before (therapeutic model) or after (prophylactic model) vaccination with a composition comprising a bacterium of the invention containing the desired tumor antigen. Vaccination with a bacterium containing the tumor antigen can be compared to control populations that are either not vaccinated, vaccinated with vehicle, or with a bacterium that expresses an irrelevant antigen. The effectiveness of the vaccine in such models can be evaluated in terms of tumor volume as a function of time after tumor injection or in terms of survival populations as a function of time after tumor injection. In one embodiment, the tumor volume in mice vaccinated with a composition comprising the bacterium is about 5%, about 10%, about 25%, about 50%, about 75%, about 90% or about 100% less than the tumor volume in mice that are either not vaccinated or are vaccinated with vehicle or a bacterium that expresses an irrelevant antigen. In another embodiment, this differential in tumor volume is observed at least about 10, about 17, or about 24 days following the implant of the tumors into the mice. In one embodiment, the median survival time in the mice vaccinated with the composition comprising a bacterium is at least about 2, about 5, about 7 or at least about 10 days longer than in mice that are either not vaccinated or are vaccinated with vehicle or bacteria that express an irrelevant antigen. 
     The host (i.e., subject) in the methods described herein, is any vertebrate, preferably a mammal, including domestic animals, sport animals, and primates, including humans. In some embodiments, the host is a mammal. In some embodiments, the host is a human. 
     The delivery of the  Listeria,  or a composition comprising the strain, may be by any suitable method, such as intradermal, subcutaneous, intraperitoneal, intravenous, intramuscular, intralymphatic, oral or intranasal, as well as by any route that is relevant for any given malignant or infectious disease or other condition. In some embodiments, the method of administration is mucosal. 
     The compositions comprising the bacteria and an immunostimulatory agent may be administered to a host simultaneously, sequentially or separately. Examples of immunostimulatory agents include, but are not limited to IL-2, IL-12, GMCSF, IL-15, B7.1, B7.2, and B7-DC and IL-14. Additional examples of stimulatory agents are provided in Section V, above 
     As used herein, an “effective amount” of a bacterium or composition (such as a pharmaceutical composition or an immunogenic composition) is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results includes results such as eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histologic and/or behavioral symptoms of a disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results includes clinical results such as inhibiting or suppressing a disease, decreasing one or more symptoms resulting from a disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of a disease, increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and/or prolonging survival of patients. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. 
     In some embodiments, for a therapeutic treatment of a cancer, an effective amount includes an amount that will result in the desired immune response, wherein the immune response either slows the growth of the targeted tumors, reduces the size of the tumors, or preferably eliminates the tumors completely. The administration of the vaccine may be repeated at appropriate intervals, and may be administered simultaneously at multiple distinct sites in the vaccinated individual. In some embodiments, for a prophylactic treatment of a cancer, an effective amount includes a dose that will result in a protective immune response such that the likelihood of an individual to develop the cancer is significantly reduced. The vaccination regimen may be comprised of a single dose, or may be repeated at suitable intervals until a protective immune response is established. 
     In some embodiments, the therapeutic treatment of an individual for cancer may be started on an individual who has been diagnosed with a cancer as an initial treatment, or may be used in combination with other treatments. For example, individuals who have had tumors surgically removed or who have been treated with radiation therapy or by chemotherapy may be treated with the vaccine in order to reduce or eliminate any residual tumors in the individual, or to reduce the risk of a recurrence of the cancer. In some embodiments, the prophylactic treatment of an individual for cancer, would be started on an individual who has an increased risk of contracting certain cancers, either due to environmental conditions or genetic predisposition. 
     The dosage of the pharmaceutical compositions or vaccines that are given to the host will vary depending on the species of the host, the size of the host, and the condition or disease of the host. The dosage of the compositions will also depend on the frequency of administration of the compositions and the route of administration. The exact dosage is chosen by the individual physician in view of the patient to be treated. 
     In some embodiments, a single dose of the pharmaceutical compositions, immunogenic compositions, or vaccines comprising the  Listeria  described herein comprises from about 10 2  to about 10 12  of the bacterial organisms. In another embodiment, a single dose comprises from about 10 5  to about 10 11  of the bacterial organisms. In another embodiment, a single dose comprises from about 10 6  to about 10 11  of the bacterial organisms. In still another embodiment, a single dose of the pharmaceutical composition or vaccine comprises from about 10 7  to about 10 10  of the bacterial organisms. In still another embodiment, a single dose of the pharmaceutical composition or vaccine comprises from about 10 7  to about 10 9  of the bacterial organisms. 
     The  Listeria  of the present invention, in some embodiments, is administered in a dose, or dosages, where each dose comprises at least about 1000  Listeria  units/kg body weight, at least about 10,000  Listeria  units/kg body weight, at least about 100,000  Listeria  units/kg body weight, at least about 1 million  Listeria  units/kg body weight, or at least about 10 million. Listeria  units/kg body weight. The present invention provides the above doses where the units of  Listeria  are colony forming units (CFU), the equivalent of CFU prior to psoralen-treatment, or where the units are number of  Listeria  cells. In some embodiments, the effective amount of attenuated  Listeria  that is measured comprises at least about 1 x 10 3  CFU/kg or at least about 1×10 3    Listeria  cells/kg. In some embodiments, the effective amount of attenuated  Listeria  that is measured comprises at least about 1×10 5  CFU/kg or at least about 1×10 5    Listeria  cells/kg. In certain embodiments, the effective amount of attenuated  Listeria  that is measured comprises at least about 1×10 6  CFU/kg or at least about 1×10 6    Listeria  cells/kg. In some embodiments, the effective amount of attenuated  Listeria  that is measured comprises at least about 1×10 7  CFU/kg or at least about 1×10 7    Listeria  cells/kg. In some further embodiments, the effective amount of attenuated  Listeria  that is measured comprises at least about 1×10 8  CFU/kg or at least about 1&gt;10 8    Listeria  cells/kg. 
     In some embodiments, a single dose of the pharmaceutical composition, immunogenic composition, or vaccine comprising the  Listeria  described herein comprises from about 1 CFU/kg to about 1×10 10  CFU/kg (CFU=colony forming units). In some embodiments, a single dose of the composition comprises from about 10 CFU/kg to about 1×10 9  CFU/kg. In another embodiment, a single dose of the composition or vaccine comprises from about 1×10 2  CFU/kg to about 1×10 8  CFU/kg. In still another embodiment, a single dose of the composition or vaccine comprises from about 1×10 3  CFU/kg to about 1×10 8  CFU/kg. In still another embodiment, a single dose of the composition or vaccine comprises from about 1×10 4  CFU/kg to about 1×10 7  CFU/kg. In some embodiments, a single dose of the composition comprises at least about 1 CFU/kg. In some embodiments, a single dose of the composition comprises at least about 10 CFU/kg. In another embodiment, a single dose of the composition or vaccine comprises at least about 1×10 2  CFU/kg. In still another embodiment, a single dose of the composition or vaccine comprises at least about 1×10 3  CFU/kg. In still another embodiment, a single dose of the composition or vaccine comprises from at least about 1×10 4  CFU/kg. 
     In some embodiments, the proper (i.e., effective) dosage amount for one host, such as human, may be extrapolated from the LD 50  data for another host, such as a mouse, using methods known to those in the art. 
     In some embodiments, the pharmaceutical composition, immunogenic composition, or vaccine comprises antigen-presenting cells, such as dendritic cells, which have been infected with the  Listeria  described herein. In some embodiments, an individual dosage of an antigen-presenting cell based vaccine comprising bacteria such as those described herein comprises between about 1×10 3  to about 1×10 10  antigen-presenting cells. In some embodiments, an individual dosage of the vaccine comprises between about 1×10 5  to about 1×10 9  antigen-presenting cells. In some embodiments, an individual dosage of the vaccine comprises between about 1×10 7  to about 1×10 9  antigen-presenting cells. 
     In some embodiments, multiple administrations of the dosage unit are preferred, either in a single day or over the course of a week or month or year or years. In some embodiments, the dosage unit is administered every day for multiple days, or once a week for multiple weeks. In some embodiments, the  Listeria  are administered to the mammalian subject at least twice, at least three times, at least four times, at least five times, at least 10 times, or at least 20 times. 
     The invention also provides a method of inducing MHC class I antigen presentation or MHC class II antigen presentation on an antigen-presenting cell comprising contacting a bacterium described herein with an antigen-presenting cell. 
     The invention further provides a method of inducing an immune response in a host to an antigen comprising, the following steps: (a) contacting a  Listeria  bacterium described herein with an antigen-presenting cell from the host, under suitable conditions and for a time sufficient to load the antigen-presenting cells; and (b) administering the antigen-presenting cell to the host. 
     VII. Kits 
     The invention further provides kits and articles of manufacture comprising the  Listeria  described herein, or compositions comprising the  Listeria  described herein. 
     Examples 
     General Methods. 
     Intracellular staining (ICS) assays involve permeabilizing the splenocytes, and treating with an antibody that binds cytokines that have accumulated inside the immune cell, where the antibody allows fluorescent tagging. Brefeldin blocks protein transport, and provokes the accumulation of cytokines within the immune cell. 
     Elispot (enzyme-linked immunospot) assays are sensitive to secreted proteins, where the proteins are secreted over a period of time from immune cells resting in a well. A capture antibody is bound to the well, which immobilized secreted cytokine. After the secretory period, the cells are removed, and a detection antibody is used to detect immobilized cytokine. The capture antibody and detection antibody bind to different regions of the cytokine. Methodological details of the ICS and elispot assays are disclosed (see, e.g., U.S. Pat. Appl. Pub. No. 2005/0249748, published Nov. 10, 2005, of Dubensky, et al.). 
     Example One 
     Schematic Diagrams Showing Transit of a Macromolecule from  Listeria  to Host Cell Cytoplasm, as Mediated by Holin or Holin Plus Lysin. Construction of Plasmids Containing Expression Cassettes Encoding Holin, Lysin, or Holin and Lysin 
     The schematic diagrams of  FIGS. 1A to 1D  disclose a number of non-limiting embodiments of the nucleic acids and  Listeria  bacteria of the invention. 
       FIG. 1A  discloses a  Listeria  bacterium harboring a plasmid. The plasmid contains a virus-derived expression cassette. Release of the plasmid from the bacterium can be mediated by holin, holin without any recombinant lysin, the combination of holin and a recombinant lysin, and the like. Once in the nucleus, transcription machinery of the host cell can use a mammalian cell compatible promoter(s), and transcribe a self-amplifying (replicating) expression cassette, where replication is mediated by proteins encoded by the expression cassette. Host cell proteins may contribute to expression and/or post-translational modification of protein encoded by the virus-derived expression cassette. The amplified mRNA encodes the desired heterologous protein, e.g., a tumor antigen or infectious agent antigen. 
     In another aspect, the plasmid that is released from the bacterium can encode a conventional transcription unit, that is, a transcription unit that is not a self-amplifying virus-derived expression cassette. 
     The dashes represent any degree of permeabilization as mediated in whole or in part by the expressed holin and/or lysin. The dashes can also represent lysis, as mediated in whole or in part by the expressed holin and/or lysin. 
       FIG. 1B  also discloses a  Listeria  bacterium harboring a plasmid. The plasmid contains a virus-derived expression cassette, where a  Listeria -compatible promoter(s) is operably linked with the expression cassette. RNA transcribed from the expression cassette is shown in the figure to be released from the bacterium, where release can be mediated by holin, holin without any recombinant lysin, the combination of holin and a recombinant lysin, and the like. 
     Once in the cytoplasm, self-amplification (replication) is mediated by proteins encoded by the expression cassette. Host cell proteins may contribute to expression and/or post-translational modification of protein encoded by the virus-derived expression cassette. The amplified mRNA encodes the desired heterologous protein, e.g., a tumor antigen or infectious agent antigen. 
       FIG. 1C  shows a  Listeria  bacterium harboring a genome-based virus-derived expression cassette, where a  Listeria -compatible promoter(s) is operably linked with the expression cassette. RNA transcribed from the expression cassette is shown in the figure to be released from the bacterium, where release can be mediated by holin, holin without any recombinant lysin, the combination of holin and a recombinant lysin, and the like. As disclosed above, once in the cytoplasm of the host cell, self-amplification occurs, where the amplified RNA encodes the desired heterologous protein(s). 
       FIG. 1D  discloses the release of polypeptides from the permeabilized or lysed  Listeria  bacterium. The polypeptide can be short peptide, a typical polypeptide of about 20-200 kD, or it can be a very large polypeptide or complex of polypeptides. In some embodiments, what is encompassed by the invention is a nucleic acid encoding the polypeptide, where there does not exist any  Listeria -compatible secretory sequence. In some embodiments, what is also encompassed is a nucleic acid encoding the polypeptide, where the polypeptide contains a  Listeria -compatible secretory sequence, and so on. 
     The figure discloses that the polypeptide is encoded by a genome-based nucleic acid, however plasmid-based nucleic acids are also contemplated. Release of the polypeptide from the bacterium can be mediated by a holin, a holin without any recombinant lysin, by the combination of holin and a recombinant lysin, and the like. 
       FIG. 2  discloses plasmids containing expression cassettes containing nucleic acids encoding holin, lysin, or holin and lysin. The nucleic acids are operably linked with actA promoter, a promoter that is specifically activated with the  Listeria  bacterium is in a host cell. The plasmids contain an attPP′ site, which is a short nucleic acid sequence used to mediate site-specific integration into a target nucleic acid, where the target nucleic acid contains a corresponding attBB′ site. Generally, the plasmid is transfected into a  Listeria  bacterium, and the target is an attBB′ site in the listerial genome. A number of attPP′ sites, corresponding attBB′ sites, and integrases that catalyze the integration, are available (see, e.g., Lauer, et al. (2002) J. Bacteriol. 183:4177-4186; ENGINEERED  LISTERIA  AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197 (filed Mar. 30, 2006), assigned to Cerus Corporation). 
     The figure also discloses that the plasmids contain a pair of loxP sites. After site-specific integration has occurred, the loxP sites can be used to mediate elimination of the intervening nucleic acid, that is, the nucleic acid encoding an antibiotic resistance gene or other selection marker gene. Cre recombinase, introduced into the bacteria by way of a plasmid, is an enzyme that recognizes loxP sites and catalyzes elimination of the nucleic acid (e.g., antibiotic gene) that is flanked by the loxP sites. 
     Assembly of the plasmids and nucleic acids of  FIG. 2  is disclosed below. 
     The polycistron containing the holin and lysin ORFs were amplified by PCR with bacteriophage PSA (Zimmer, et al. (2003) Mol. Microbiol. 50, 303-317) genomic DNA as template and using the following primers: 
     
       
         
           
               
            
               
                 PL529 (forward): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 23) 
               
            
           
           
               
            
               
                 5′ tt GGATTCP atgaaaattaactggaaagt 3′ 
               
               
                   
               
               
                 PL530 (reverse): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 24) 
               
            
           
           
               
            
               
                 5′ ttGAGCTCGGCCGCGGCCGCagtatgaggaagtggaacgt 3′ 
               
            
           
         
       
     
     After amplification, the PCR product was purified over a Qiagen® column, eluted and digested with ClaI and EagI. After complete digestion, the fragment was cloned into pINT (ENGINEERED  LISTERIA  AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197, filed Mar. 30, 2006, and assigned to Cerus Corporation) cut with the same set of restriction enzymes downstream of the actA promoter, resulting in pBHE292. 
     The plasmid expressing lysin only was engineered in a similar manner. The lysin ORF was PCR amplified with PSA genomic DNA as template using the following primers: 
     
       
         
           
               
            
               
                 PL612 (forward): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 25) 
               
            
           
           
               
            
               
                 5′ AAAATCGATATGATAGTAATGAGTAATTATAGTATGTCG 3′ 
               
               
                   
               
               
                 PL613 (reverse): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 26) 
               
            
           
           
               
            
               
                 5′ AAAGCGGCCGCAGTATGAGGAAGTGGAACGTATGTACTTAT 3′ 
               
            
           
         
       
     
     After amplification, the PCR products was purified over a Qiagen® column, eluted and digested with ClaI and EagI. The fragment was cloned into pINT downstream of the actA promoter resulting in pBHE361. 
     The plasmid expressing holin only was derived from pBHE292 by deletion of the lysin sequence from the unique NruI site to the unique NotI. After restriction digest, the plasmid was blunted using T4 ploymerase, purified over a Qiagen® column and self ligated. This resulted in pBHE340. 
     After engineering the three plasmids and confirming their fidelity by sequence analysis, they were integrated at the tRNA Arg  locus in the genome of selected  Listeria  strains using previously described methods (Lauer, et al. (2002) J. Bacteriol. 184, 4177-4186). Integration was confirmed on erythromycin resistant  Listeria  colonies by PCR with NC16 (5′ gtcaaaacatacgctcttatc 3′) and PL95 (5′ acataatcagtccaaagtagatgc 3′). 
     Example Two  
     Derivation of Recombinant  L. monocytogenes  (Lm) Strains Containing Holin, Lysin or Holin and Lysin Expression Cassettes and Characterization of their Growth Properties in Broth and in Mammalian Host Cells 
       Listeria monocytogenes  (Lm) was engineered to contain a nucleic acid encoding a listeriophage holin (PSA phage), listeriophage lysin (PSA phage), or both holin and lysin. Site-specific integration was at the attBB′ site naturally occurring at the tRNA Arg  locus of the listerial genome, were transcription was controlled by the listerial actA promoter, a promoter specifically activated by conditions inside a host cell. 
       FIG. 3A  demonstrates that growth of the parental Lm strain (“CRS-100”) and of Lm-holin-lysin were the same in broth. Bacterial viability of LmΔactAΔinlB (“CRS-100”) and BH226 was determined at 7 hrs and 24 hrs and was comparable at both time points for both  Listeria  strains (˜5×10 9  cfu/ml). Therefore, the holin-lysin cassette is not detrimental to the growth of the bacteria in broth culture. The identical growth curves demonstrate lack of expression of holin and lysin with culture in broth, even though Lm-holin-lysin was engineered to contain nucleic acids encoding these two proteins. BH226 is attenuated for growth in mammalian host cells compared to the parental  Listeria  strain ( FIG. 3B ). 
       FIG. 3C  is a schematic diagram of the listerial constructs containing a polypeptide comprising a nucleic acid encoding holin, a nucleic acid encoding lysin, or nucleic acids encoding holin and lysin. Expression was from the actA promoter. The parental Lm strain, “CRS-100,” is Lm ΔactAΔinlB, a strain of Lm that is attenuated by deletions in the actA gene and inlB gene.  FIG. 3D  demonstrates that expression of both holin and lysin (BH276) are required to inhibit intracellular growth of  L. monocytogenes  in mammalian cells (J774 cells). In contrast,  L. monocytogenes  strains that express only holin (BH334) or only lysin (BH336) are not inhibited in intracellular bacterial growth in J774 cells as compared to wild-type  L. monocytogenes  (DP14056). 
       FIG. 3E  demonstrates that expression of only holin (and with no expression of lysin) does not necessarily impair growth of Lm when Lm is grown inside host mammalian cells (J774 cells). The figure discloses near-identical intracellular growth curves of Lm-holin and parental Lm, and somewhat lesser growth of a listerial construct expressing two copies of the holin gene (Lm-holin-holin). One copy of holin does not affect growth or bacterial viability in host cells (BH567), whereas two copies of the actA promoter-holin cassette does affect the growth and viability of the bacteria in host cells (BH727).  FIG. 3F , repeat of growth attenuation of holin+lysin containing bacteria where growth attenuation is dramatic. Repeated experiments demonstrated that Lm-holin showed no consistent difference in intracellular growth, when compared with that of a control parental Lm not containing a nucleic acid encoding holin. In other words, the holin expressed by Lm-holin did not inhibit intracellular growth. 
     The invention contemplates a Lm-holin capable of sustained growth and/or metabolism, where the sustained growth and/or metabolism results in continued expression of nucleic acids encoding a heterologous antigen. While not limiting the invention to any particular property or advantage, a contemplated advantage is as follows. Lm-holin containing a nucleic acid encoding an antigen, or a nucleic acid encoding a viral-derived expression cassette, is contemplated to show sustained expression of the nucleic acid, while the expressed holin mediates transit of the expressed antigen (or the expressed viral-derived expression cassette) from the bacterium into the host cell&#39;s cytosol. 
       FIG. 3F  discloses lesser growth properties of Lm expressing both holin and lysin (Lm-holin-lysin; Lm-holy). The figure discloses a rapid rate of disappearance of viable bacteria, in the case of Lm-holin-lysin and, in contrast, steady growth of the parental  Listeria  strain. 
       FIG. 4  provides photographs where bacteria, actin, and dsDNA were visualized by anti-Lm antibodies, anti-actin antibodies, and diamidinophenylindole (DAPI), respectively. DAPI staining results in visualization of bacteria by way of staining the bacterial genome, and it also visualizes the mammalian host cell&#39;s nucleus. DAPI and anti-0 antigen antibodies are described (see, e.g., (see, e.g., Hazeleger, et al. (2006) Int. J. Food Microbiol. June 23 epub; Aarnisalo, et al. (2003) J. Food Prot. 66:249-255). Anti-actin antibodies for staining are available (see, e.g., Amersham Pharmacia Biotech, Piscataway, NJ; Sigma Aldrich, St. Louis, Mo.). 
     The figure demonstrates that expression of both holin and lysin by Lm-holin-lysin results in fragmentation of the  Listeria  bacteria, failure to show the expected actin trails. Fragmentation was demonstrated with the anti-O antigen antibodies, which stain an extracellular marker on  Listeria.  Fragmentation and destruction of the bacteria was confirmed by DAPIs failure to stain bacteria. In contrast, parental Lm, Lm-lysin, and Lm-holin, showed equivalent staining by anti-O antigen antibodies. 
     These results, which show that holin alone did not fragment bacteria under the conditions of the experiment, are consistent with those shown above, demonstrating that parental Lm and Lm-holin can show similar or identical growth rates. 
     The failure of lysin alone, as expressed by Lm-lysin, to fragment the bacteria suggests that Lm-lysin cannot serve as a suitable vehicle for mediating transfer of nucleic acids, viral-based expression cassettes, polypeptides, from the inside of a  Listeria  bacterium to an external environment, e.g., a host cell cytoplasm. 
     In other words, the immunofluorescence images of J774 cells infected with various  Listeria  strains show the following. For the three strains DP-L4056, BH334, and BH336, the bacteria appear as wild type—that is, intact and associated with actin tails. In the case of BH276, while some bacteria are associated with actin tails, there are several instances of “exploded bacteria”, where the anti- Listeria  antibody recognizes fragments of bacteria. This is visual confirmation of the growth curve data. 
     Further details for EXAMPLE TWO were as follows. 
     Strains were constructed by conjugation from  E. coli  to  L. monocytogenes  essentially as described (Lauer, et al. (2002) J. Bacteriol. 184:4177-4186). Holin and/or lysin cassettes were introduced to wild-type (DP-L4056), ΔactA, ΔuvrAB, or ΔactAΔinlB strain backgrounds. 
     Growth curves were done in broth culture starting from a stationary phase overnight 3 ml VPP (Veggie Peptone Phosphate, Oxoid). A 1:100 dilution was done in VPP or YNG (Yeast no glucose) media and OD 600 nm readings were taken at 30 to 60 minute time intervals. Viable bacteria in a culture were determined by plating appropriate serial dilutions on VPP plates and counting colonies. 
     Growth curves in J774 cells were done essentially as described (Portnoy, et al. (1988) J. Exp. Med. 167:1459-1471). Briefly, J774 cells were maintained in DMEM+10% FBS and antibiotic and seeded in either 6 well dishes with 12 mm coverslips or into 24 well plates without coverslips at a density of 1e6 cells per ml. The next day, cells were washed with PBS, resuspended in media without antibiotic, and infected with a fresh 30 ° C. overnight of the appropriate  Listeria  strain at a multiplicity of infection (moi) of 1, 2, 5, or 10. After 30 or 60 minutes, cells were washed with PBS and media was replaced with DMEM containing 50 μg/ml of Gentamycin. Growth was monitored by sampling at various time points in triplicate by lysing host cells in water and plating appropriate dilutions on VPP. 
     Immunofluorescence was done essentially as described (Skoble, et al. (2000) J. Cell Biol. 150:527-538). Infections were performed as above on 12 mm coverslips, and cells were fixed in 3.5% formaldehyde. Bacteria were stained with anti-listeria O antigen polycolonal antibody (Difco) and visualized with a secondary antibody conjugated to FITC. Actin was stained with Rhodamine phalloidin. DAPI stained the nuclei of host cells and DNA of the bacteria and was present in the mounting media (Molecular Probes, Eugene, Oreg.). 
     Example Three 
     Utility of Recombinant  L. monocytogenes  (Lm) Strains Expressing Holin, Lysin, or Holin and Lysin, to Deliver a Eukaryotic Expression Plasmid to the Cytoplasm of a Host Cell 
       FIG. 5A  is a schematic diagram showing a plasmid, suitable for placing in a  Listeria  bacterium, for subsequent release, and for monitoring release by way of activity of expressed luciferase. The plasmid encodes luciferase operably linked with a promoter compatible with mammalian host cell transcription machinery. 
       FIG. 5B  compares release of the luciferase-encoding plasmid from  Listeria  to the cytoplasm of BHK cells. Luciferase-catalyzed luminescence generated by infecting host cells with  Listeria  monocytogenes (Lm) constructs containing DNA plasmid encoding luciferase. Lm constructs were parental Lm (no plasmid), parental Lm (+plasmid); Lm-lysin (+plasmid); Lm-holin-lysin (+plasmid); and Lm-holin (+plasmid). Luciferase-catalyzed luminescence, as shown by luminescence counts per second (LCPS), was determined under two conditions, that is, where the indicated Lm was added to 1×10 5  BHK cells. 
     The figure demonstrates that lysin alone, as expressed by Lm-lysin, failed to stimulate release of plasmid from the bacterium, as compared to the control bacterium containing the plasmid but no holin and no lysin. In contrast, holin alone, as expressed by Lm-holin, produced significant luminescence, for example, 7,500 counts per second. The combination of holin and lysin, as expressed by Lm-holin-lysin, also produced significant luminescence. 
     To conclude, expression of holin alone, or holin with lysin, resulted in readily measurable release of the plasmid from the bacterium, while expression of lysin alone did not result in any detectable release. 
     These results are consistent with those noted above in the bacterial fragmentation experiments. Failure of lysin alone, as expressed by Lm-lysin, to release luciferase-plasmid from  Listeria monocytogenes  suggests that Lm-lysin cannot serve as a suitable vehicle for mediating transfer of nucleic acids, viral-based expression cassettes, polypeptides, and the like, from the inside of a  Listeria  bacterium to an external environment, e.g., to a mammalian host cell cytoplasm. 
     Construction of the relevant plasmids, nucleic acids, and  Listeria  strains is disclosed below. 
     A plasmid was constructed allowing maintenance in  Listeria  as well as expression of luciferase in host cells. The backbone of this plasmid was derived from pAM401 (Wirth, et al. (1986) J. Bacteriol. 165:831-836). In order to make this plasmid conducive to bacterial conjugation, the oriT from pPL2 (Lauer, et al. (2002) J. Bacteriol. 184:4177-4186) was amplified by PCR and cloned into the unique SacII site resulting in pAM401oriT. The initial luciferase plasmid was constructed by digesting pAM401oriT with EagI and BamHI, treating with CIP (NEB) and purifying over a Qiagen® column. The luciferase cassette was obtained by digesting pGL3 control vector (Promega, Madison, Wis.) with NotI and BamHI and gel purifying the 2609 by fragment. The two fragments were ligated together using T4 DNA ligase (NEB) and colonies confirmed by PCR and restriction enzyme digest. This resulted in the mammalian expression vector pBHE539. After initial experiments demonstrated lower than desired luminescence values, the SV40 promoter was replaced with a region of the pRL-CMV vector (Promega, Madison, Wis.) containing the CMV enhancer and immediate early promoter as well as a chimeric intron. The plasmid pBHE539 was digested with BglII and StuI, treated with CIP and cleaned up over a Qiagen® column. The CMV promoter region was obtained by digesting pRL-CMV with BglII and ScaI and gel purifying the 1035 by fragment. The fragments were ligated together using T4 DNA ligase and clones confirmed by PCR and restriction digest resulting in pBHE573. When compared in mammalian cell infection experiments, pBHE573 resulted in luminescence values 10-20 fold higher than pBHE539. 
     To test the utility of the various holin and/or lysin expressing  Listeria  strains for delivery of a eukaryotic expression plasmid to host cells, the vector pBHE573 was moved into the various holin/lysin strains by conjugation as described previously (Lauer, et al. (2002) J. Bacteriol. 184, 4177-4186). The day before cell infection, strains harboring the plasmid were grown in BHI medium+10 ug/ml chloramphenicol (for maintenance of plasmid) at 30° C. for 16 hrs. BHK cells (ref) were maintained in a T75 flask containing 15 mls DMEM+10% FBS+1xNEAA+50 mg/L penicillin-streptomycin at 37° C/5% CO 2 . Cells at circa 90% confluence were treated with 3 mL trypsin solution for 15 minutes at 37° C. Cells were then diluted with 10 mls DMEM+10% FBS+1xNEAA, pelleted and resuspended in 3 mls DMEM+10% FBS+1xNEAA. After cell number was determined, culture was diluted to 3 E 5  cells/ml and transferred to 12-well plates (1 ml culture/well) for overnight incubation at 37° C/5% CO 2  (results in one cell doubling). The day of infection, one ml of bacterial culture at a concentration of 2 E9 bacteria/ml was pelleted and resuspended in 1 ml DPBS (Mediatech) for each strain. This bacterial suspension was used to inoculate DMEM+10% FBS+1xNEAA to a final concentration of 6 E 7  bacteria/ml (MOI of 100). Medium was removed from the BHK cells by aspiration and replaced with 1 ml of bacterial suspension. Cells were infected for 1 hour at 37° C./5% CO 2  After infection, the medium was removed by aspiration and replaced with DMEM+10% FBS+1xNEAA+50 mg/L gentamicin. Cells were then incubated overnight at 37° C./5% CO 2 . After 24 hrs, cells were assayed for luciferase activity using the Steady-Glo Luciferase Assay System (Promega, Madison, Wis.). Medium was removed by aspiration and replaced with 220 ul cell lysis buffer containing luciferin. After incubation of cells for 10 mins at RT, two 100 ul aliquots/well were transferred to a 96-well plate for reading in a luminometer (Perkin-Elmer Trilux). 
     Example Four  
     Utility of Recombinant  L. monocytogenes  (Lm) Strains Expressing Holin, Lysin, or Holin and Lysin, for Delivering a Plasmid DNA Replicon to the Cytoplasm of a Mammalian Host Cell 
       FIG. 6A  discloses a plasmid, pSH263, containing a nucleic acid that is an alphavirus-based expression cassette, where the expression cassette contains a nucleic acid encoding β-galactosidase (lacZ gene). The non-structural genes encoded by the expression cassette mediate self-amplification (replication) of the plasmid, where replication of the expression cassette occurs after the plasmid leaves the  Listeria  bacterium for the mammalian host cell&#39;s cytosol. 
       FIGS. 6B-6E  disclose biological results where the indicated Lm constructs were administered to mammalian cells. The mammalian cells were BHK cells or J774 cells, as indicated. As indicated, the Lm constructs were parental Lm (Lm wild-type); Lm holin-lysin (Lm-HoLy); Lm-lysin; and Lm-holin. “Lm-Ho-Ho” means  Listeria monocytogenes  containing two copies of a nucleic acid encoding holin. 
     Other listerial constructs were based on KBMA Lm ΔuvrAB, a preparation of  Listeria monocytogenes  mutated in a DNA repair gene (uvrAB) and treated with an agent (psoralen) that cross-links the listerial genome. KBMA Lm ΔuvrAB is metabolically active however its genome contains a small number of chemical cross-links, and the bacterium is metabolically active but is not able to form colonies (see, e.g., Brockstedt, et al. (2005) Nature Medicine 11:853-860; U.S. Pub. No. US 2004/0197343 of Dubensky, et al.). The KBMA-based constructs included KBMA Lm ΔuvrAB-holin-lysin (KBMA Lm ΔuvrAB-HoLy). 
       FIG. 6B  shows light microscope pictures of BHK cells treated with the indicated vector. BHK cells were infected with  Listeria  containing a plasmid encoding an alphavirus-derived expression cassette. β-galactosidase expression was measured in BHK cells at 24 hr. following introduction of pSH263 plasmid alphavirus expression cassette (pAM402oriT pSin-lacZ). A positive signal (dark spots) indicates expression of β-galactosidase (Panels A-F). 
     Panel A discloses that transfecting BHK cells with the plasmid (virus-based replicon) resulted in a high degree of expression. (Here, the plasmid was not inside a  Listeria  bacterium. 
     Panel B shows that β-galactosidase expression from a different type of plasmid, pBHE530 ( FIG. 6A ), a plasmid where expression is controlled by a conventional promoter (CMV promoter). (This plasmid does not contain a viral-based replicon, and there is no self-amplification of message in the cytoplasm.) Expression was low. 
     Panel C shows expression using wild type Lm (no holin; no lysin) containing the plasmid encoding the virus-based replicon. β-galactosidase expression was low. 
     Panel D shows expression using Lm-lysin containing the virus-based replicon. Expression was also low. The low degree of expression using Lm-lysin, a degree similar to that of parental Lm (no holin; no lysin), demonstrates that lysin alone does not mediate release of a plasmid from Lm. In other words, β-galactosidase expression from Lm-lysin was similar to background. 
     Panel E demonstrates that expression using Lm-holin-lysin containing the virus-based replicon results in a relatively high degree of β-galactosidase expression. Panel F demonstrates that similar high degrees of β-galactosidase expression were found where Lm-holin was used. To summarize, various Lm constructs were administered to BHK cells, where the Lm contained a plasmid bearing a self-amplifying virus-based expression cassette. Where the Lm construct was Lm-holin or Lm-holin-lysin, the expressed holin mediated release of the plasmid out of the bacterium to the host cell&#39;s cytosol, where plasmid-encoded enzymes could amplify the expression cassette, and where the mammalian translational machinery could express large amounts of heterologous antigen (β-galactosidase). The results showed that lysin alone did not mediate plasmid release. 
       FIG. 6C  discloses a similar experiment using BHK cells, as disclosed by photographs of BHK cells containing various Lm constructs, and  FIG. 6D  discloses histograms quantitating the raw data. As indicated, BHK cells were treated with buffer only (mock); parental Lm (DP-L4056); Lm-holin; Lm-holin-lysin (Lm-HoLy); and Lm-holin-holin (Lm-Ho-Ho). 
       FIG. 6E  shows a similar experiment as above, but with J774 cells instead of BHK cells. The bacterial constructs were: live Lm ΔuvrAB-holin-lysin (Lm ΔuvrAB-HoLy); KBMA Lm ΔuvrAB-holin-lysin (Lm ΔuvrAB-HoLy); KBMA Lm ΔuvrAB; and live Lm-holin-lysin (Lm-HoLy). The term “live” means that psoralen had not been added to convert the “live” bacteria to killed but metabolically active (KBMA) bacteria. The fluorescent photomicrographs demonstrate the fragmentation of the  Listeria  bacteria, where the bacteria expressed both holin and lysin. The light microscope photographs reveals the signal generated by β-galactosidase biosynthesized in the cytoplasm of the host J774 cells, where the β-galactosidase is encoded by the plasmid DNA replicon. β-galactosidase activity, which is dependent on both release of the plasmid from the bacterium, amplification in the J774 cell&#39;s cytosol, and translation, was greatest with live Lm-holin-lysin and with Lm ΔuvrAB-holin-lysin. Significant β-galactosidase activity was also found with KBMA Lm ΔuvrAB-holin-lysin. 
     In other words, the top photographs show two examples of KBMA bacteria lysing within the host cell in a Holin-Lysin dependent manner. For each panel, the left is stained with the anti- Listeria  polyclonal antibody and visualized with FITC. Bacteria that are lysin are noted with an arrow in each panel. The right is stained with rhodamine-phalloidin. The bottom photographs show the following. Holin-Lysin KBMA  Listeria  bacteria are able to deliver to Sindbis virus-lacZ replicon to BHK. The first and last panel are live Holin-Lysin control strains, panel two is KBMA holin-lysin with the replicon, panel three is KBMA without holin-lysin. 
     To conclude, the data demonstrates the utility of a virus-derived expression cassette, as provided by Lm-holin vector, in expressing a heterologous antigen. 
     Details for EXAMPLE FOUR were as follows. Construction of pSH263 was as described. The shuttle plasmid pSH263 was constructed from a Sindbis viral replicon and a lacZ reporter cloned downstream of an RSV promoter for intracellular DNA launch. The Sindbis virus replicon, lacZ and the RSV promoter components of pSH263 were derived from the plasmids Sinrep/lacZ and Sinrep21 from Dr. Sondra Schlesinger of Washington University School of Medicine, St. Louis, Mo. A BamHI-EagI fragment containing the RSV promoter and a portion of nonstructural proteins was cut from Sinrep21 and inserted into BamHI-EagI sites of pSH252 to create the plasmid pSH258. The remaining nonstructural proteins and a lacZ reporter were cut from Sinrep/lacZ as a BglII-NotI fragment and cloned into the BglII-EagI sites of pSH258. The resulting plasmid, pSH263 was used to transform  E. coli  strain SM10 resulting in strain B-Ec-266. The plasmid pSH263 was transferred from B-Ec-266 to various  Listeria  strains by conjugation. The construction of pSH263 was confirmed by EcoRI digestion and the integrity of the replicon confirmed by transient transfection of BHK cells followed by staining for β-galactosidase activity as described below. 
     The plasmid pSH252 was derived from pAM401 (Wirth, et al. (1986) J. Bacteriol. 165:831-836) by inserting the oriT from pINT into the SacII site of pAM401. The oriT from pINT was amplified with the following primers that include SacII sites (underlined) using Platinum Pfx polymerase according to product instructions: 
     
       
         
           
               
               
            
               
                   
                 WL214 
               
            
           
           
               
            
               
                 (SEQ ID NO: 27) 
               
            
           
           
               
               
            
               
                   
                 5′ acat CCGCGG TTTCAGTGCAATTTATCTCTTCAAATG 3′ 
               
               
                   
                   
               
               
                   
                 WL215 
               
            
           
           
               
            
               
                 (SEQ ID NO: 28) 
               
            
           
           
               
               
            
               
                   
                 5′ atct CCGCGG ATGTATGCTATACGAAGTTATGCG 3′ 
               
            
           
         
       
     
     The resulting PCR product was digested with SacII and inserted into SacII-digested pAM401. The resulting plasmid, pSH252, replicates as an episome in both  E. coli  and  Listeria  hosts and is transferred from  E. coli  donors to  Listeria  hosts by conjugation as described previously. 
     As a positive control for DNA delivery to eukaryotic cells by  Listeria,  the strain BH276 (pBHE530) was derived. This strain expresses holin and lysin, and contains the plasmid pBHE530. 
     The plasmid pBHE530 was constructed as follows. As a positive control for DNA delivery to eukaryotic cells by  Listeria,  the strain BH276(pBHE530) was derived. This strain expresses holin and lysin and contains the plasmid pBHE530. The plasmid pBHE530 was constructed by subcloning the CMV immediate-early promoter and lacZ gene from the plasmid pShuttle-CMV-lacZ (Stratagene, San Diego, Calif.) into the plasmid pSH252. The CMV promoter-lacZ expression cassette was removed from pShuttle-CMV-lacZ by SapI/SacII digestion, gel purified and blunted with T4 DNA polymerase. The 3473 bp fragment was inserted into the EcoRV site of pSH252 to complete construction of pBHE530. The plasmid pBHE530 was used to transform  E. coli  SM10 and the  Listeria  strain BH276(pBHE530) completed by subsequent conjugation. 
     Transient transfections in BHK cells were performed to confirm that the Sindbis virus replicon could be launched from pSH263 in eukaryotic cells. BHK cells were seeded in 6-well plates at a density of 5×10 5  cells/well in complete growth medium (DMEM contianing 10% fetal calf serum and non-essential amino acids) and cultured overnight at 37° C. Prior to transfection, monolayers were 90-100% confluent and washed twice in complete growth medium. Four micrograms of pSH263 DNA were diluted in 250 uL OptiMEM (Invitrogen Corp.) and mixed with 20 μL Lipofectamine 2000® (Invitrogen Corp.) diluted in 250 μL OptiMEM according to product instructions. Following a 20 minute incubation at room temperature, 500 μL DNA-lipofectamine mixture was added directly to each well of BHK cells in 2 ml complete grown medium. Transfected cells were cultured at 37° C. and stained for β-galactosidase activity at 24 hours post-transfection. 
     β-Galactosidase activity staining was performed using a procedure modified from Sanes, et al. (1986) EMBO J. 5:3133-3142, and published on the Invitrogen web site. Briefly, transfected or infected BHK cells were washed with PBS and fixed lightly in 2% (v/v) formaldehyde, 0.2% (v/v) glutaraldehyde in PBS for 5 minutes at room temperature, washed with PBS then stained overnight with 1 mg/mL X-gal in 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride and 1× PBS. After staining cells were rinsed with PBS and fixed for 10 minutes at room temperature in 10% (v/v) formaldehyde in PBS. Fixed cells were washed once with PBS then stored at 4° C. in PBS until analysis. 
     In order to investigate the ability of  Listeria  strains expressing holin, lysin, or holin and lysin to deliver Sindbis virus replicons to infected BHK cells, the plasmid pSH263 was introduced into the  Listeria  strains DP-L4056, BH334, BH336, BH276 and BH727 by conjugation with the  E. coli  donor strain B-Ec-266 resulting in the strains B-Lm-274, B-Lm-277, B-Lm-276, B-Lm-278 and B-Lm-414 respectively. As a positive control for DNA delivery to BHK cells, an SM10 strain containing pBHE530 was conjugated with the  Listeria  strain BH276 to make BH276(pBHE530). Six-well plates were seeded with 4×10 5  BHK/well and 96-well plates seeded with 2×10 4  cell/well in complete growth medium and cultured at 37° C. overnight. All  Listeria  strains were cultured in BHI containing 10 ug/mL chloramphenicol overnight at 30° C. without shaking. Overnight  Listeria  cultures were washed twice with PBS and used to infect BHK cells at a multiplicity of infection (MOI) of 200 in 500 uL serum-free DMEM. After 1 hour at 37° C., inocula were aspirated and cells washed three times in serum-free DMEM containing 50 ug/mL gentamycin. Infected cells were cultured in complete growth medium with 50 ug/mL gentamycin and stained for β-galactosidase activity at 24 hours post-transfection. 
     An experiment with Killed But Metabolically Active  Listeria monocytogenes  (KBMA Lm) was conducted as follows. To investigate the ability of photochemically inactivated  Listeria  to deliver pSH263 to eukaryotic cells, the following strains were made. The uvrAB locus was deleted from the  Listeria  strain DP-L4029 resulting in 4029uvr.  Listeria  strain 4029uvr was conjugated with  E. coli  SM10 harboring the plasmid pBHE292 resulting the  Listeria  strain B-Lm-284. B-Lm-284 contains both holin and lysin downstream of the actA promoter integrated at the tRNA-Arg locus. Next, pSH263 was transferred into B-Lm-284 and 4029uvr by conjugation with B-Ec-266 resulting in the strains B-Lm-288 and B-Lm-289, respectively. Photochemical inactivation of  Listeria  containing the uvrAB deletion has been described previously. Briefly, overnight cultures of B-Lm-288 and B-Lm-289 were grown at 37° C. in filter-sterilized BHI. Overnight cultures were diluted 1:50 in fresh filter-sterilized BHI and incubated at 37° C. at 300 ron to OD 600 =0.5. A 50 ml aliquot of each culture was transferred to a fresh glass flask and S-59 was added to a concentration of 200 nM and grown at 37° C. at 300 rpm for one hour to reach an approximately OD 600 =1.0. Cultures were transferred to a 100 mm polystyrene petri dish and UVA irradiated at 6 J/cm 2 . S-59 treated  Listeria  were collected by centrifugation at 2300×g for 20 minutes at 4° C. and washed once with 50 mL PBS. The final pellet was suspended in PBS and used immediately to infect BHK cells. Inactivation was confirmed by plating serial dilutions of  Listeria  cultures on non-selective medium before and after UVA irradiation. 
     Six-well plates were seeded with 4×10 5  BHK/well and cultured overnight at 37° C. Cells were infected at MOI of 200 and 400 with inactivated B-Lm-288 and B-Lm-289 as described earlier. Infected BHK were cultured 24 hours at 37° C. in complete medium containing 50 ug/mL gentamycin then stained for β-galactosidase activity as described above. 
     The sequence of pSH263 is shown below. The sequence contains an RSV promoter, non-structural proteins 1-4 (nsp104), and lacZ reporter sequence: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 29) 
                   
               
            
           
           
               
               
            
               
                 ggatccagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacat 
                   
               
               
                   
               
               
                 gccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcg 
               
               
                   
               
               
                 tgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattccgca 
               
               
                   
               
               
                 ttgcagagatattgtatttaagtgccctacctcgataccgtcgagattgacggcgtagtaca 
               
               
                   
               
               
                 cactattgaatcaaacagccgaccaattgcactaccatcacaatggagaagccagtagtaaa 
               
               
                   
               
               
                 cgtagacgtagacccccagagtccgtttgtcgtgcaactgcaaaaaagcttcccgcaatttg 
               
               
                   
               
               
                 aggtagtagcacagcaggtcactccaaatgaccatgctaatgccagagcattttcgcatctg 
               
               
                   
               
               
                 gccagtaaactaatcgagctggaggttcctaccacagcgacgatcttggacataggcagcgc 
               
               
                   
               
               
                 accggctcgtagaatgttttccgagcaccagtatcattgtgtctgccccatgcgtagtccag 
               
               
                   
               
               
                 aagacccggaccgcatgatgaaatacgccagtaaactggcggaaaaagcgtgcaagattaca 
               
               
                   
               
               
                 aacaagaacttgcatgagaagattaaggatctccggaccgtacttgatacgccggatgctga 
               
               
                   
               
               
                 aacaccatcgctctgctttcacaacgatgttacctgcaacatgcgtgccgaatattccgtca 
               
               
                   
               
               
                 tgcaggacgtgtatatcaacgctcccggaactatctatcatcaggctatgaaaggcgtgcgg 
               
               
                   
               
               
                 accctgtactggattggcttcgacaccacccagttcatgttctcggctatggcaggttcgta 
               
               
                   
               
               
                 ccctgcgtacaacaccaactgggccgacgagaaagtccttgaagcgcgtaacatcggacttt 
               
               
                   
               
               
                 gcagcacaaagctgagtgaaggtaggacaggaaaattgtcgataatgaggaagaaggagttg 
               
               
                   
               
               
                 aagcccgggtcgcgggtttatttctccgtaggatcgacactttatccagaacacagagccag 
               
               
                   
               
               
                 cttgcagagctggcatcttccatcggtgttccacttgaatggaaagcagtcgtacacttgcc 
               
               
                   
               
               
                 gctgtgatacagtggtgagttgcgaaggctacgtagtgaagaaaatcaccatcagtcccggg 
               
               
                   
               
               
                 atcacgggagaaaccgtgggatacgcggttacacacaatagcgagggcttcttgctatgcaa 
               
               
                   
               
               
                 agttactgacacagtaaaaggagaacgggtatcgttccctgtgtgcacgtacatcccggcca 
               
               
                   
               
               
                 ccatatgcgatcagatgactggtataatggccacggatatatcacctgacgatgcacaaaaa 
               
               
                   
               
               
                 cttctggttgggctcaaccagcgaattgtcattaacggtaggactaacaggaacaccaacac 
               
               
                   
               
               
                 catgcaaaattaccttctgccgatcatagcacaagggttcagcaaatgggctaaggagcgca 
               
               
                   
               
               
                 aggatgatcttgataacgagaaaatgctgggtactagagaacgcaagcttacgtatggctgc 
               
               
                   
               
               
                 ttgtgggcgtttcgcactaagaaagtacattcgttttatcgcccacctggaacgcagacctg 
               
               
                   
               
               
                 cgtaaaagtcccagcctcttttagcgcttttcccatgtcgtccgtatggacgacctctttgc 
               
               
                   
               
               
                 ccatgtcgctgaggcagaaattgaaactggcattgcaaccaaagaaggaggaaaaactgctg 
               
               
                   
               
               
                 caggtctcggaggaattagtcatggaggccaaggctgcttttgaggatgctcaggaggaagc 
               
               
                   
               
               
                 cagagcggagaagctccgagaagcacttccaccattagtggcagacaaaggcatcgaggcag 
               
               
                   
               
               
                 ccgcagaagttgtctgcgaagtggaggggctccaggcggacatcggagcagcattagttgaa 
               
               
                   
               
               
                 accccgcgcggtcacgtaaggataatacctcaagcaaatgaccgtatgatcggacagtatat 
               
               
                   
               
               
                 cgttgtctcgccaaactctgtgctgaagaatgccaaactcgcaccagcgcacccgctagcag 
               
               
                   
               
               
                 atcaggttaagatcataacacactccggaagatcaggaaggtacgcggtcgaaccatacgac 
               
               
                   
               
               
                 gctaaagtactgatgccagcaggaggtgccgtaccatggccagaattcctagcactgagtga 
               
               
                   
               
               
                 gagcgccacgttagtgtacaacgaaagagagtttgtgaaccgcaaactataccacattgcca 
               
               
                   
               
               
                 tgcatggccccgccaagaatacagaagaggagcagtacaaggttacaaaggcagagcttgca 
               
               
                   
               
               
                 gaaacagagtacgtgtttgacgtggacaagaagcgttgcgttaagaaggaagaagcctcagg 
               
               
                   
               
               
                 tctggtcctctcgggagaactgaccaaccctccctatcatgagctagctctggagggactga 
               
               
                   
               
               
                 agacccgacctgcggtcccgtacaaggtcgaaacaataggagtgataggcacaccggggtcg 
               
               
                   
               
               
                 ggcaagtcagctattatcaagtcaactgtcacggcacgagatcttgttaccagcggaaagaa 
               
               
                   
               
               
                 agaaaattgtcgcgaaattgaggccgacgtgctaagactgaggggtatgcagattacgtcga 
               
               
                   
               
               
                 agacagtagattcggttatgctcaacggatgccacaaagccgtagaagtgctgtacgttgac 
               
               
                   
               
               
                 gaagcgttcgcgtgccacgcaggagcactacttgccttgattgctatcgtcaggccccgcaa 
               
               
                   
               
               
                 gaaggtagtactatgcggagaccccatgcaatgcggattcttcaacatgatgcaactaaagg 
               
               
                   
               
               
                 tacatttcaatcaccctgaaaaagacatatgcaccaagacattctacaagtatatctcccgg 
               
               
                   
               
               
                 cgttgcacacagccagttacagctattgtatcgacactgcattacgatggaaagatgaaaac 
               
               
                   
               
               
                 cacgaacccgtgcaagaagaacattgaaatcgatattacaggggccacaaagccgaagccag 
               
               
                   
               
               
                 gggatatcatcctgacatgtttccgcgggtgggttaagcaattgcaaatcgactatcccgga 
               
               
                   
               
               
                 catgaagtaatgacagccgcggcctcacaagggctaaccagaaaaggagtgtatgccgtccg 
               
               
                   
               
               
                 gcaaaaagtcaatgaaaacccactgtacgcgatcacatcagagcatgtgaacgtgttgctca 
               
               
                   
               
               
                 cccgcactgaggacaggctagtgtggaaaaccttgcagggcgacccatggattaagcagccc 
               
               
                   
               
               
                 actaacatacctaaaggaaactttcaggctactatagaggactgggaagctgaacacaaggg 
               
               
                   
               
               
                 aataattgctgcaataaacagccccactccccgtgccaatccgttcagctgcaagaccaacg 
               
               
                   
               
               
                 tttgctgggcgaaagcattggaaccgatactagccacggccggtatcgtacttaccggttgc 
               
               
                   
               
               
                 cagtggagcgaactgttcccacagtttgcggatgacaaaccacattcggccatttacgcctt 
               
               
                   
               
               
                 agacgtaatttgcattaagtttttcggcatggacttgacaagcggactgttttctaaacaga 
               
               
                   
               
               
                 gcatcccactaacgtaccatcccgccgattcagcgaggccggtagctcattgggacaacagc 
               
               
                   
               
               
                 ccaggaacccgcaagtatgggtacgatcacgccattgccgccgaactctcccgtagatttcc 
               
               
                   
               
               
                 ggtgttccagctagctgggaagggcacacaacttgatttgcagacggggagaaccagagtta 
               
               
                   
               
               
                 tctctgcacagcataacctggtcccggtgaaccgcaatcttcctcacgccttagtccccgag 
               
               
                   
               
               
                 tacaaggagaagcaacccggcccggtcaaaaaattcttgaaccagttcaaacaccactcagt 
               
               
                   
               
               
                 acttgtggtatcagaggaaaaaattgaagctccccgtaagagaatcgaatggatcgccccga 
               
               
                   
               
               
                 ttggcatagccggtgcagataagaactacaacctggctttcgggtttccgccgcaggcacgg 
               
               
                   
               
               
                 tacgacctggtgttcatcaacattggaactaaatacagaaaccaccactttcagcagtgcga 
               
               
                   
               
               
                 agaccatgcggcgaccttaaaaaccctttcgcgttcggccctgaattgccttaacccaggag 
               
               
                   
               
               
                 gcaccctcgtggtgaagtcctatggctacgccgaccgcaacagtgaggacgtagtcaccgct 
               
               
                   
               
               
                 cttgccagaaagtttgtcagggtgtctgcagcgagaccagattgtgtctcaagcaatacaga 
               
               
                   
               
               
                 aatgtacctgattttccgacaactagacaacagccgtacacggcaattcaccccgcaccatc 
               
               
                   
               
               
                 tgaattgcgtgatttcgtccgtgtatgagggtacaagagatggagttggagccgcgccgtca 
               
               
                   
               
               
                 taccgcaccaaaagggagaatattgctgactgtcaagaggaagcagttgtcaacgcagccaa 
               
               
                   
               
               
                 tccgctgggtagaccaggcgaaggagtctgccgtgccatctataaacgttggccgaccagtt 
               
               
                   
               
               
                 ttaccgattcagccacggagacaggcaccgcaagaatgactgtgtgcctaggaaagaaagtg 
               
               
                   
               
               
                 atccacgcggtcggccctgatttccggaagcacccagaagcagaagccttgaaattgctaca 
               
               
                   
               
               
                 aaacgcctaccatgcagtggcagacttagtaaatgaacataacatcaagtctgtcgccattc 
               
               
                   
               
               
                 cactgctatctacaggcatttacgcagccggaaaagaccgccttgaagtatcacttaactgc 
               
               
                   
               
               
                 ttgacaaccgcgctagacagaactgacgcggacgtaaccatctattgcctggataagaagtg 
               
               
                   
               
               
                 gaaggaaagaatcgacgcggcactccaacttaaggagtctgtaacagagctgaaggatgaag 
               
               
                   
               
               
                 atatggagatcgacgatgagttagtatggattcatccagacagttgcttgaagggaagaaag 
               
               
                   
               
               
                 ggattcagtactacaaaaggaaaattgtattcgtacttcgaaggcaccaaattccatcaagc 
               
               
                   
               
               
                 agcaaaagacatggcggagataaaggtcctgttccctaatgaccaggaaagtaatgaacaac 
               
               
                   
               
               
                 tgtgtgcctacatattgggtgagaccatggaagcaatccgcgaaaagtgcccggtcgaccat 
               
               
                   
               
               
                 aacccgtcgtctagcccgcccaaaacgttgccgtgcctttgcatgtatgccatgacgccaga 
               
               
                   
               
               
                 aagggtccacagacttagaagcaataacgtcaaagaagttacagtatgctcctccacccccc 
               
               
                   
               
               
                 ttcctaagcacaaaattaagaatgttcagaaggttcagtgcacgaaagtagtcctgtttaat 
               
               
                   
               
               
                 ccgcacactcccgcattcgttcccgcccgtaagtacatagaagtgccagaacagcctaccgc 
               
               
                   
               
               
                 tcctcctgcacaggccgaggaggcccccgaagttgtagcgacaccgtcaccatctacagctg 
               
               
                   
               
               
                 ataacacctcgcttgatgtcacagacatctcactggatatggatgacagtagcgaaggctca 
               
               
                   
               
               
                 cttttttcgagctttagcggatcggacaactctattactagtatggacagttggtcgtcagg 
               
               
                   
               
               
                 acctagttcactagagatagtagaccgaaggcaggtggtggtggctgacgttcatgccgtcc 
               
               
                   
               
               
                 aagagcctgcccctattccaccgccaaggctaaagaagatggcccgcctggcagcggcaaga 
               
               
                   
               
               
                 aaagagcccactccaccggcaagcaatagctctgagtccctccacctctcttttggtggggt 
               
               
                   
               
               
                 atccatgtccctcggatcaattttcgacggagagacggcccgccaggcagcggtacaacccc 
               
               
                   
               
               
                 tggcaacaggccccacggatgtgcctatgtctttcggatcgttttccgacggagagattgat 
               
               
                   
               
               
                 gagctgagccgcagagtaactgagtccgaacccgtcctgtttggatcatttgaaccgggcga 
               
               
                   
               
               
                 agtgaactcaattatatcgtcccgatcagccgtatcttttccactacgcaagcagagacgta 
               
               
                   
               
               
                 gacgcaggagcaggaggactgaatactgactaaccggggtaggtgggtacatattttcgacg 
               
               
                   
               
               
                 gacacaggccctgggcacttgcaaaagaagtccgttctgcagaaccagcttacagaaccgac 
               
               
                   
               
               
                 cttggagcgcaatgtcctggaaagaattcatgccccggtgctcgacacgtcgaaagaggaac 
               
               
                   
               
               
                 aactcaaactcaggtaccagatgatgcccaccgaagccaacaaaagtaggtaccagtctcgt 
               
               
                   
               
               
                 aaagtagaaaatcagaaagccataaccactgagcgactactgtcaggactacgactgtataa 
               
               
                   
               
               
                 ctctgccacagatcagccagaatgctataagatcacctatccgaaaccattgtactccagta 
               
               
                   
               
               
                 gcgtaccggcgaactactccgatccacagttcgctgtagctgtctgtaacaactatctgcat 
               
               
                   
               
               
                 gagaactatccgacagtagcatcttatcagattactgacgagtacgatgcttacttggatat 
               
               
                   
               
               
                 ggtagacgggacagtcgcctgcctggatactgcaaccttctgccccgctaagcttagaagtt 
               
               
                   
               
               
                 acccgaaaaaacatgagtatagagccccgaatatccgcagtgcggttccatcagcgatgcag 
               
               
                   
               
               
                 aacacgctacaaaatgtgctcattgccgcaactaaaagaaattgcaacgtcacgcagatgcg 
               
               
                   
               
               
                 tgaactgccaacactggactcagcgacattcaatgtcgaatgctttcgaaaatatgcatgta 
               
               
                   
               
               
                 atgacgagtattgggaggagttcgctcggaagccaattaggattaccactgagtttgtcacc 
               
               
                   
               
               
                 gcatatgtagctagactgaaaggccctaaggccgccgcactatttgcaaagacgtataattt 
               
               
                   
               
               
                 ggtcccattgcaagaagtgcctatggatagattcgtcatggacatgaaaagagacgtgaaag 
               
               
                   
               
               
                 ttacaccaggcacgaaacacacagaagaaagaccgaaagtacaagtgatacaagccgcagaa 
               
               
                   
               
               
                 cccctggcgactgcttacttatgcgggattcaccgggaattagtgcgtaggcttacggccgt 
               
               
                   
               
               
                 cttgcttccaaacattcacacgctttttgacatgtcggcggaggattttgatgcaatcatag 
               
               
                   
               
               
                 cagaacacttcaagcaaggcgacccggtactggagacggatatcgcatcattcgacaaaagc 
               
               
                   
               
               
                 caagacgacgctatggcgttaaccggtctgatgatcttggaggacctgggtgtggatcaacc 
               
               
                   
               
               
                 actactcgacttgatcgagtgcgcctttggagaaatatcatccacccatctacctacgggta 
               
               
                   
               
               
                 ctcgttttaaattcggggcgatgatgaaatccggaatgttcctcacactttttgtcaacaca 
               
               
                   
               
               
                 gttttgaatgtcgttatcgccagcagagtactagaagagcggcttaaaacgtccagatgtgc 
               
               
                   
               
               
                 agcgttcattggcgacgacaacatcatacatggagtagtatctgacaaagaaatggctgaga 
               
               
                   
               
               
                 ggtgcgccacctggctcaacatggaggttaagatcatcgacgcagtcatcggtgagagacca 
               
               
                   
               
               
                 ccttacttctgcggcggatttatcttgcaagattcggttacttccacagcgtgccgcgtggc 
               
               
                   
               
               
                 ggatcccctgaaaaggctgtttaagttgggtaaaccgctcccagccgacgacgagcaagacg 
               
               
                   
               
               
                 aagacagaagacgcgctctgctagatgaaacaaaggcgtggtttagagtaggtataacaggc 
               
               
                   
               
               
                 actttagcagtggccgtgacgacccggtatgaggtagacaatattacacctgtcctactggc 
               
               
                   
               
               
                 attgagaacttttgcccagagcaaaagagcattccaagccatcagaggggaaataaagcatc 
               
               
                   
               
               
                 tctacggtggtcctaaatagtcagcatagtacatttcatctgactaatactacaacaccacc 
               
               
                   
               
               
                 acctctagaccatggatcccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc 
               
               
                   
               
               
                 caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccg 
               
               
                   
               
               
                 caccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttc 
               
               
                   
               
               
                 cggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtc 
               
               
                   
               
               
                 gtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtaaccta 
               
               
                   
               
               
                 tcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctca 
               
               
                   
               
               
                 catttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgtt 
               
               
                   
               
               
                 aactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgttt 
               
               
                   
               
               
                 gccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatgg 
               
               
                   
               
               
                 tgctgcgttggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcatt 
               
               
                   
               
               
                 ttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccac 
               
               
                   
               
               
                 tcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagt 
               
               
                   
               
               
                 tgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggc 
               
               
                   
               
               
                 accgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacact 
               
               
                   
               
               
                 acgtctcaaggtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcgg 
               
               
                   
               
               
                 tggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttc 
               
               
                   
               
               
                 cgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgagg 
               
               
                   
               
               
                 cgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgc 
               
               
                   
               
               
                 aggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaac 
               
               
                   
               
               
                 catccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatat 
               
               
                   
               
               
                 tgaaacccacggcatggtgccaatcaatcgtctgaccgatgatccgcgctggctaccggcga 
               
               
                   
               
               
                 tgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctgg 
               
               
                   
               
               
                 tcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatc 
               
               
                   
               
               
                 tgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgata 
               
               
                   
               
               
                 ttatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatgg 
               
               
                   
               
               
                 tccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgc 
               
               
                   
               
               
                 ccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatc 
               
               
                   
               
               
                 cccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaa 
               
               
                   
               
               
                 aacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagtt 
               
               
                   
               
               
                 ctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaac 
               
               
                   
               
               
                 accagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatac 
               
               
                   
               
               
                 ctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgct 
               
               
                   
               
               
                 ggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctg 
               
               
                   
               
               
                 aactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaac 
               
               
                   
               
               
                 gcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaa 
               
               
                   
               
               
                 cctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatgg 
               
               
                   
               
               
                 atttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttca 
               
               
                   
               
               
                 cagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgc 
               
               
                   
               
               
                 accgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcg 
               
               
                   
               
               
                 aacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagat 
               
               
                   
               
               
                 acacttgctgatgcggtgctgattacgaccggtcacgcgtggcagcatcaggggaaaacctt 
               
               
                   
               
               
                 atttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatg 
               
               
                   
               
               
                 ttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcag 
               
               
                   
               
               
                 gtagcagagcgggtaaactgqctcggattagggccgcaagaaaactatcccgaccgccttac 
               
               
                   
               
               
                 tgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcc 
               
               
                   
               
               
                 cgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgc 
               
               
                   
               
               
                 ggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcg 
               
               
                   
               
               
                 ccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattg 
               
               
                   
               
               
                 gtggcgacgactcctggagcccgtcagtatcggcggaattcagctgagcgccgttcgctacc 
               
               
                   
               
               
                 attaccagttggtctggtgtcaaaaataataataaccgggcaggggggatcctagacgctac 
               
               
                   
               
               
                 gccccaatgatccgaccagcaaaactcgatgtacttccgaggaactgatgtgcataatgcag 
               
               
                   
               
               
                 gaattcgatatcaagctagcatgcaggccttgggcccaatgatccgaccagcaaaactcgat 
               
               
                   
               
               
                 gtacttccgaggaactgatgtgcataatgcatcaggctggtacattagatccccgcttaccg 
               
               
                   
               
               
                 cgggcaatatagcaacactaaaaactcgatgtacttccgaggaagcgcagtgcataatgctg 
               
               
                   
               
               
                 cgcagtgttgccacataaccactatattaaccatttatctagcggacgccaaaaactcaatg 
               
               
                   
               
               
                 tatttctgaggaagcgtggtgcataatgccacgcagcgtctgcataacttttattatttctt 
               
               
                   
               
               
                 ttattaatcaacaaaattttgtttttaacatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaa 
               
               
                   
               
               
                 aaaaaaaaagggaattcctcgattaattaagcggccg 
               
            
           
         
       
     
     Example Five 
     Utility of Recombinant  L. monocytogenes  (Lm) Strains Expressing Holin, Lysin, or Holin and Lysin, for Delivering Messenger RNA (mRNA) to the Cytoplasm of a Mammalian Host Cell 
     Plasmids encoding an IRES sequence operably linked with a nucleic acid encoding luciferase were transfected into  Listeria monocytogenes  (Lm), as described below. The Lm, in turn, were used to infect mammalian cells. In the first study ( FIG. 7 ), the plasmid encoded luciferase. 
     Details of the above work are as follows. For  FIG. 7 , luminiscence from luciferase activity was a measure of transcription of the RNA in the Lm, release of the RNA to the cytoplasm of the mammalian host cell, and translation of the message. The IRES sequence of the mRNA mediates translation and can substitute, for example, in whole or in part, for a cap sequence on the mRNA.  FIG. 7  demonstrates that Lm-holin (BH743) mediated significant release of the mRNA from the bacterium to the mammalian host cell&#39;s cytoplasm. Here, luminescence reached about 1.2 LCPS units. Lm-holin-lysin (4056holy) also mediated significant release of the mRNA, where luminescence ranged from 0.3-0.8 LCPS units. 
     In order to determine the utility of delivering RNA from  Listeria  to infected host cells, a plasmid was constructed with the luciferase ORF under the control of the actA promoter and containing an intervening IRES sequence. This plasmid allows the transcription of the luciferase message in  Listeria,  the holin-mediated secretion of the message and subsequent translation in the host cell cytoplasm. 
     The construction of the plasmid was accomplished by PCR amplifying both a synthetic IRES fragment (ClaI/BamHI ends) and the luciferase ORF from pGL3 control vector (Promega, BamHI/NotI ends). Both PCR products were purified over a Qiagen® column and digested with the appropriate restriction enzymes. The vector pBHE135 was digested with ClaI and NotI, treated with CIP and purified over a Qiagen® column. The vector and two fragments were ligated together using T4 DNA ligase (NEB) in a three-way ligation. Chloramphenicol resistant colonies were screened by colony PCR and confirmed by restriction digest. This plasmid was introduced into  Listeria  by conjugation (Lauer, et al. (2002) J. Bacteriol. 184:4177-4186), selected on erythromycin and integration confirmed by PCR, resulting in BH721. This strain was cured of the vector backbone sequences (ENGINEERED  LISTERIA  AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197, filed Mar. 30, 2006, and assigned to Cerus Corporation) resulting in erythromycin sensitive colonies (BH741). This strain was subsequently conjugated with SM10 cells harboring either pBHE633 (actAp_holin directed to comK) or pBHE636 (actAp_holin-lysin directed to comK) resulting in the  Listeria monocytogenes  strains, BH743 and BH745, respectively. 
       Listeria monocytogenes  was introduced into BHK cells as follows. These strains along with DP-L4056 were grown overnight in BHI medium at 30° C. BHK cells were plated at an initial density of 2 E 5  cells/ml in 12-well plates. The following day 1 ml of bacterial culture was pelleted and resuspended in 1 ml DPBS. This suspension was used to inoculate DMEM+10% FBS+1xNEAA to a final density of 8×10 7  bacteria/ml. 
     Medium was removed from BHK cells by aspiration and the cells were inoculated with the bacteria-containing medium (MOI 200). After a 1 hour infection period at 37° C., medium was replaced with DMEM+10% FBS+1xNEAA+50 gentamicin. Cells were assayed for luciferase activity several times over the course of 48 hours as described in Example 3. 
     Example Six 
     Utility of Recombinant  L. monocytogenes  (Lm) Strains Expressing Holin, Lysin, or Holin and Lysin for Delivering a Cap-Independent, Viral-Based Replicon to the Cytoplasm of a Mammalian Host Cell 
     The present invention, in certain aspects, provides a  Listeria  bacterium encompassing a nucleic acid containing a virus-derived expression cassette, where the expression cassette encodes the following RNA embodiments. In one embodiment, the RNA does not contain an IRES. Alternatively, the expression cassette is derived from a viral genome that does not contain an IRES (e.g., Yellow fever virus; alphavirus), and an IRES is engineered into the RNA. 
     In another embodiment, the expression cassette is derived from a viral genome that has a naturally occurring IRES (e.g., Picornaviruses), where this IRES is maintained in the expression cassette. In still another aspect, the expression cassette is derived from a viral genome having a naturally occurring IRES, where this IRES is deleted or inactivated. Moreover, what is also supplied is an expression cassette derived from a viral genome having an IRES, but where the IRES is deleted or inactivated and replaced with a different IRES. 
     The invention includes  Listeria,  nucleic acids, and methods, where the RNA initially made in the  Listeria  bacterium is functional in the host cell&#39;s cytoplasm, even when the RNA is uncapped. What is also included are  Listeria,  nucleic acids, and methods, where the RNA is functional in the host cell&#39;s cytoplasm, even when the RNA is uncapped, and where subsequent capping increases function, and also where subsequent capping does not increase function. 
       FIG. 8A  is a schematic diagram showing replication of the viral-based expression cassette, as it can occur in a mammalian host cell&#39;s cytoplasm, as well as biosynthesis of capped subgenomic RNA, where the capped subgenomic RNA is used for expression of a heterologous polypeptide, for example, a tumor antigen or infectious agent antigen. 
     In one aspect of the present invention, recombinant  L. monocytogenes  strains expressing holin, lysin, or holin and lysin that in addition encode a viral-based self-amplifying RNA, also known as a replicon, are provided. In such embodiments, the viral-based replicon can be encoded by a plasmid DNA or, alternatively, can be integrated into the bacterial chromosome at any preferred site by non-limiting alternative methods including homologous recombination or by site-specific integration vectors. Replicons derived from viruses having single-stranded RNA genomes of positive polarity, that are encoded by the recombinant  L. monocytogenes  strains expressing holin, lysin, or holin and lysin are provided. In one embodiment, the replicon is a component of a eukaryotic expression cassette that is functionally linked to a DNA polymerase II (pol II) promoter, including as non-limiting examples the cytomegalovirus (CMV) immediate early or Rous sarcoma virus (RSV) promoters, and encoded by a plasmid DNA. As a non-limiting example, the replicon can be derived from any member of the Togaviridae family, including Sindbis virus (SIN), or Venezuelan equine encephalitis virus (VEE). In this embodiment, the SIN or VEE replicons are deleted of most of their genomes encoding the viral structural proteins (sPs), rendering these expresssed RNA molecules incapable of producing productively infectious virus. While replicons derived from SIN are described, in can be appreciated by those skilled in the art that such embodiments can be derived from any member of the Togaviridae family. As a non-limiting example, replicon compositions containing a heterologous gene of interest, such as a gene encoding a desired tumor antigen, in place of the structural proteins (sPs) are provided. In the described invention, the plasmid encoding the replicon is released into the cytoplasm of infected mammalian host cells in culture or in an intact vaccinated animal following infection with a recombinant  L. monocytogenes  strain expressing holin, lysin, or holin and lysin. Within this context, following migration to the nucleus and synthesis of the replicon RNA initiated from the pol II promoter, the replicon is transported to the cytoplasm, where the nonstructural proteins (nsPs), or replicase, are first translated by the host cell machinery, which in turn program the amplification of the RNA replicon, by ordered steps including first a synthesis of a full-length replicon-complementary strand of negative polarity, which in turn serves as template for the synthesis of additional replicon full-length RNA molecules and also for the synthesis of subgenomic RNA molecules, through initiation from an internal promoter that is functional only when the RNA is of negative polarity. The subgenomic RNA is the translational template for the encoded gene of interest, and is synthesized in molar excess as compared to the level of full-length RNA replicon molecules synthesized. 
     In some embodiments of the invention, replicons that are derived from cap-independent viruses having single-stranded RNA genomes of positive polarity, or from viruses having single-stranded RNA genomes of positive polarity that are further modified such that they are cap-independent, are described. Such native or modified virus derived replicons are cap-independent by virtue of containing a functional internal ribosomal entry site (IRES). Such cap-independent replicons can be encoded by a plasmid DNA or, alternatively, can be integrated into the bacterial chromosome of recombinant  L. monocytogenes  strains that in addition express holin, lysin, or holin and lysin. In this aspect of the invention, the cap-independent replicon RNA is functionally linked to a bacterial promoter, as a non-limiting example a PrfA-inducible promoter such as actA. Synthesis of the cap-independent replicon RNA by the bacterium is induced in the cytoplasm of infected mammalian host cells in culture or in an intact vaccinated animal following infection with a recombinant  L. monocytogenes  strain, and is released into the mammalian host cell by expression of holin, lysin, or holin and lysin. Subsequently, the nsPs are translated from the cap-independent replicon RNA by the host cell machinery, resulting in self-amplification of the replicon and protein synthesis of the encoded heterologous gene, as described above. 
     A. Construction of Cap-Independent Alphavirus Replicons. 
     Construction of Cap-Independent Sindbis Virus Replicon (pCO390): 
     A DNA fragment containing the following ordered elements was synthesized by DNA2.0 (Menlo Park, Calif.): a sp6 promoter, sindbis virus tRNA Asp  defective-interferring (DI) 5′terminus, UTR and codons 1-40 of nsp1 followed by the internal ribosomal entry site (IRES) from ECMV and codons 1-40 of nsp1 with alternate codon useage and received on the plasmid pJ10:4934. The insert from pJ10:4394 was fused with the Sindbis virus replicon from the plasmid Sinrep/lacZ using SOE-PCR. Briefly, the insert from pJ10:4394 was amplified with the primers: 
     
       
         
           
               
            
               
                 PL860 
               
            
           
           
               
            
               
                 (SEQ ID NO: 30) 
               
            
           
           
               
            
               
                 5′ ATGGAAAAACGCCAGCAACGCGAGCTCGTATGGACATATTGTCGT 
               
               
                 TAGAACG 3′ 
               
               
                   
               
               
                 PL861 
               
            
           
           
               
            
               
                 (SEQ ID NO: 31) 
               
            
           
           
               
            
               
                 5′ CCTCCAGCTCGATTAGTTTACTGGCCAGGTGGCTGAAGGCTCT 
               
               
                 TG 3′ 
               
            
           
         
       
     
     Using the plasmid Sinrep/LacZ as template, a portion of the Sinbis virus replicon was amplified with the following primers: 
     
       
         
           
               
            
               
                 PL862 
               
            
           
           
               
            
               
                 (SEQ ID NO: 32) 
               
            
           
           
               
            
               
                 5′ GAGCCTTCAGCCACCTGGCCAGTAAACTAATCGAGCTGGAGGTT 
               
               
                 CC 3′ 
               
               
                   
               
               
                 WL224 
               
            
           
           
               
            
               
                 (SEQ ID NO: 33) 
               
            
           
           
               
            
               
                 5′ ATACCGGCCGTGGCTAGTATC 3′ 
               
            
           
         
       
     
     Products from these two primary reactions were pooled and used as template for a secondary amplification with the primer set PL860/WL224. Phusion polymerase® (New England Biolabs, Beverly, Mass.) was used in all amplifications. The secondary PCR product was digested with Sac I and Eag I and inserted into the Sac I and Eag I sites of pBluescript KS+® (Stratagene) and designated pCO330. The fidelity of the insert was verified by sequence analysis. The remainder of the Sindbis virus replicon and lacZ reporter was isolated from a partial Eag I digestion of the plasmid Sinrep/LacZ. Following gel purification, the 8055 bp Eag I fragment was inserted into the Eag I site of pCO330 resulting in the plasmid pCO390. 
     Shown below is the DNA sequence of the Sindbis virus replicon from plasmid pCO390. The plasmid contains an Sp6 promoter, tRNA end, nsp1 codons 1-40, IRES, nsp1 (1-40) wobble codons, the rest of nsp1-4, lacZ, through EagI restriction site. 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 34) 
                   
               
            
           
           
               
               
            
               
                 atttaggggacactatagggatatagtggtgagtatccccgcctgtcacgcgggagaccggg 
                   
               
               
                   
               
               
                 gttcggttccccgacggggagccaaacagccgaccaattgcactaccatcacaatggagaag 
               
               
                   
               
               
                 ccagtagtaaacgtagacgtagacccccagagtccgtttgtcgtgcaactgcaaaaaagctt 
               
               
                   
               
               
                 cccgcaatttgaggtagtagcacagcaggtcactccaaatgaccatgctaatgccagagcat 
               
               
                   
               
               
                 tttcgcatctggcgcatgcatctagggcggccaattccgcccctctccctccccccccccta 
               
               
                   
               
               
                 acgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgtgattttcc 
               
               
                   
               
               
                 accatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgag 
               
               
                   
               
               
                 cattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaagg 
               
               
                   
               
               
                 aagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcag 
               
               
                   
               
               
                 cggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacc 
               
               
                   
               
               
                 tgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaat 
               
               
                   
               
               
                 ggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatg 
               
               
                   
               
               
                 ggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacg 
               
               
                   
               
               
                 tctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataagcttatg 
               
               
                   
               
               
                 gaaaaaccggtggtcaatgtggatgtcgatccacaaagcccattcgtagtacagcttcagaa 
               
               
                   
               
               
                 gtcatttccacagttcgaagtggtcgcccagcaagtaaccccgaacgaccacgccaacgcaa 
               
               
                   
               
               
                 gagccttcagccacctggccagtaaactaatcgagctggaggttcctaccacagcgacgatc 
               
               
                   
               
               
                 ttggacataggcagcgcaccggctcgtagaatgttttccgagcaccagtatcattgtgtctg 
               
               
                   
               
               
                 ccccatgcgtagtccagaagacccggaccgcatgatgaaatacgccagtaaactggcggaaa 
               
               
                   
               
               
                 aagcgtgcaagattacaaacaagaacttgcatgagaagattaaggatctccggaccgtactt 
               
               
                   
               
               
                 gatacgccggatgctgaaacaccatcgctctgctttcacaacgatgttacctgcaacatgcg 
               
               
                   
               
               
                 tgccgaatattccgtcatgcaggacgtgtatatcaacgctcccggaactatctatcatcagg 
               
               
                   
               
               
                 ctatgaaaggcgtgcggaccctgtactggattggcttcgacaccacccagttcatgttctcg 
               
               
                   
               
               
                 gctatggcaggttcgtaccctgcgtacaacaccaactgggccgacgagaaagtccttgaagc 
               
               
                   
               
               
                 gcgtaacatcggactttgcagcacaaagctgagtgaaggtaggacaggaaaattgtcgataa 
               
               
                   
               
               
                 tgaggaagaaggagttgaagcccgggtcgcgggtttatttctccgtaggatcgacactttat 
               
               
                   
               
               
                 ccagaacacagagccagcttgcagagctggcatcttccatcggtgttccacttgaatggaaa 
               
               
                   
               
               
                 gcagtcgtacacttgccgctgtgatacagtggtgagttgcgaaggctacgtagtgaagaaaa 
               
               
                   
               
               
                 tcaccatcagtcccgggatcacgggagaaaccgtgggatacgcggttacacacaatagcgag 
               
               
                   
               
               
                 ggcttcttgctatgcaaagttactgacacagtaaaaggagaacgggtatcgttccctgtgtg 
               
               
                   
               
               
                 cacgtacatcccggccaccatatgcgatcagatgactggtataatggccacggatatatcac 
               
               
                   
               
               
                 ctgacgatgcacaaaaacttctggttgggctcaaccagcgaattgtcattaacggtaggact 
               
               
                   
               
               
                 aacaggaacaccaacaccatgcaaaattaccttctgccgatcatagcacaagggttcagcaa 
               
               
                   
               
               
                 atgggctaaggagcgcaaggatgatcttgataacgagaaaatgctgggtactagagaacgca 
               
               
                   
               
               
                 agcttacgtatggctgcttgtgggcgtttcgcactaagaaagtacattcgttttatcgccca 
               
               
                   
               
               
                 cctggaacgcagacctgcgtaaaagtcccagcctcttttagcgcttttcccatgtcgtccgt 
               
               
                   
               
               
                 atggacgacctctttgcccatgtcgctgaggcagaaattgaaactggcattgcaaccaaaga 
               
               
                   
               
               
                 aggaggaaaaactgctgcaggtctcggaggaattagtcatggaggccaaggctgcttttgag 
               
               
                   
               
               
                 gatgctcaggaggaagccagagcggagaagctccgagaagcacttccaccattagtggcaga 
               
               
                   
               
               
                 caaaggcatcgaggcagccgcagaagttgtctgcgaagtggaggggctccaggcggacatcg 
               
               
                   
               
               
                 gagcagcattagttgaaaccccgcgcggtcacgtaaggataatacctcaagcaaatgaccgt 
               
               
                   
               
               
                 atgatcggacagtatatcgttgtctcgccaaactctgtgctgaagaatgccaaactcgcacc 
               
               
                   
               
               
                 agcgcacccgctagcagatcaggttaagatcataacacactccggaagatcaggaaggtacg 
               
               
                   
               
               
                 cggtcgaaccatacgacgctaaagtactgatgccagcaggaggtgccgtaccatggccagaa 
               
               
                   
               
               
                 ttcctagcactgagtgagagcgccacgttagtgtacaacgaaagagagtttgtgaaccgcaa 
               
               
                   
               
               
                 actataccacattgccatgcatggccccgccaagaatacagaagaggagcagtacaaggtta 
               
               
                   
               
               
                 caaaggcagagcttgcagaaacagagtacgtgtttgacgtggacaagaagcgttgcgttaag 
               
               
                   
               
               
                 aaggaagaagcctcaggtctggtcctctcgggagaactgaccaaccctccctatcatgagct 
               
               
                   
               
               
                 agctctggagggactgaagacccgacctgcggtcccgtacaaggtcgaaacaataggagtga 
               
               
                   
               
               
                 taggcacaccggggtcgggcaagtcagctattatcaagtcaactgtcacggcacgagatctt 
               
               
                   
               
               
                 gttaccagcggaaagaaagaaaattgtcgcgaaattgaggccgacgtgctaagactgagggg 
               
               
                   
               
               
                 tatgcagattacgtcgaagacagtagattcggttatgctcaacggatgccacaaagccgtag 
               
               
                   
               
               
                 aagtgctgtacgttgacgaagcgttcgcgtgccacgcaggagcactacttgccttgattgct 
               
               
                   
               
               
                 atcgtcaggccccgcaagaaggtagtactatgcggagaccccatgcaatgcggattcttcaa 
               
               
                   
               
               
                 catgatgcaactaaaggtacatttcaatcaccctgaaaaagacatatgcaccaagacattct 
               
               
                   
               
               
                 acaagtatatctcccggcgttgcacacagccagttacagctattgtatcgacactgcattac 
               
               
                   
               
               
                 gatggaaagatgaaaaccacgaacccgtgcaagaagaacattgaaatcgatattacaggggc 
               
               
                   
               
               
                 cacaaagccgaagccaggggatatcatcctgacatgtttccgcgggtgggttaagcaattgc 
               
               
                   
               
               
                 aaatcgactatcccggacatgaagtaatgacagccgcggcctcacaagggctaaccagaaaa 
               
               
                   
               
               
                 ggagtgtatgccgtccggcaaaaagtcaatgaaaacccactgtacgcgatcacatcagagca 
               
               
                   
               
               
                 tgtgaacgtgttgctcacccgcactgaggacaggctagtgtggaaaaccttgcagggcgacc 
               
               
                   
               
               
                 catggattaagcagcccactaacatacctaaaggaaactttcaggctactatagaggactgg 
               
               
                   
               
               
                 gaagctgaacacaagggaataattgctgcaataaacagccccactccccgtgccaatccgtt 
               
               
                   
               
               
                 cagctgcaagaccaacgtttgctgggcgaaagcattggaaccgatactagccacggccggta 
               
               
                   
               
               
                 tcgtacttaccggttgccagtggagcgaactgttcccacagtttgcggatgacaaaccacat 
               
               
                   
               
               
                 tcggccatttacgccttagacgtaatttgcattaagtttttcggcatggacttgacaagcgg 
               
               
                   
               
               
                 actgttttctaaacagagcatcccactaacgtaccatcccgccgattcagcgaggccggtag 
               
               
                   
               
               
                 ctcattgggacaacagcccaggaacccgcaagtatgggtacgatcacgccattgccgccgaa 
               
               
                   
               
               
                 ctctcccgtagatttccggtgttccagctagctgggaagggcacacaacttgatttgcagac 
               
               
                   
               
               
                 ggggagaaccagagttatctctgcacagcataacctggtcccggtgaaccgcaatcttcctc 
               
               
                   
               
               
                 acgccttagtccccgagtacaaggagaagcaacccggcccggtcaaaaaattcttgaaccag 
               
               
                   
               
               
                 ttcaaacaccactcagtacttgtggtatcagaggaaaaaattgaagctccccgtaagagaat 
               
               
                   
               
               
                 cgaatggatcgccccgattggcatagccggtgcagataagaactacaacctggctttcgggt 
               
               
                   
               
               
                 ttccgccgcaggcacggtacgacctggtgttcatcaacattggaactaaatacagaaaccac 
               
               
                   
               
               
                 cactttcagcagtgcgaagaccatgcggcgaccttaaaaaccctttcgcgttcggccctgaa 
               
               
                   
               
               
                 ttgccttaacccaggaggcaccctcgtggtgaagtcctatggctacgccgaccgcaacagtg 
               
               
                   
               
               
                 aggacgtagtcaccgctcttgccagaaagtttgtcagggtgtctgcagcgagaccagattgt 
               
               
                   
               
               
                 gtctcaagcaatacagaaatgtacctgattttccgacaactagacaacagccgtacacggca 
               
               
                   
               
               
                 attcaccccgcaccatctgaattgcgtgatttcgtccgtgtatgagggtacaagagatggag 
               
               
                   
               
               
                 ttggagccgcgccgtcataccgcaccaaaagggagaatattgctgactgtcaagaggaagca 
               
               
                   
               
               
                 gttgtcaacgcagccaatccgctgggtagaccaggcgaaggagtctgccgtgccatctataa 
               
               
                   
               
               
                 acgttggccgaccagttttaccgattcagccacggagacaggcaccgcaagaatgactgtgt 
               
               
                   
               
               
                 gcctaggaaagaaagtgatccacgcggtcggccctgatttccggaagcacccagaagcagaa 
               
               
                   
               
               
                 gccttgaaattgctacaaaacgcctaccatgcagtggcagacttagtaaatgaacataacat 
               
               
                   
               
               
                 caagtctgtcgccattccactgctatctacaggcatttacgcagccggaaaagaccgccttg 
               
               
                   
               
               
                 aagtatcacttaactgcttgacaaccgcgctagacagaactgacgcggacgtaaccatctat 
               
               
                   
               
               
                 tgcctggataagaagtggaaggaaagaatcgacgcggcactccaacttaaggagtctgtaac 
               
               
                   
               
               
                 agagctgaaggatgaagatatggagatcgacgatgagttagtatggattcatccagacagtt 
               
               
                   
               
               
                 gcttgaagggaagaaagggattcagtactacaaaaggaaaattgtattcgtacttcgaaggc 
               
               
                   
               
               
                 accaaattccatcaagcagcaaaagacatggcggagataaaggtcctgttccctaatgacca 
               
               
                   
               
               
                 ggaaagtaatgaacaactgtgtgcctacatattgggtgagaccatggaagcaatccgcgaaa 
               
               
                   
               
               
                 agtgcccggtcgaccataacccgtcgtctagcccgcccaaaacgttgccgtgcctttgcatg 
               
               
                   
               
               
                 tatgccatgacgccagaaagggtccacagacttagaagcaataacgtcaaagaagttacagt 
               
               
                   
               
               
                 atgctcctccaccccccttcctaagcacaaaattaagaatgttcagaaggttcagtgcacga 
               
               
                   
               
               
                 aagtagtcctgtttaatccgcacactcccgcattcgttcccgcccgtaagtacatagaagtg 
               
               
                   
               
               
                 ccagaacagcctaccgctcctcctgcacaggccgaggaggcccccgaagttgtagcgacacc 
               
               
                   
               
               
                 gtcaccatctacagctgataacacctcgcttgatgtcacagacatctcactggatatggatg 
               
               
                   
               
               
                 acagtagcgaaggctcacttttttcgagctttagcggatcggacaactctattactagtatg 
               
               
                   
               
               
                 gacagttggtcgtcaggacctagttcactagagatagtagaccgaaggcaggtggtggtggc 
               
               
                   
               
               
                 tgacgttcatgccgtccaagagcctgcccctattccaccgccaaggctaaagaagatggccc 
               
               
                   
               
               
                 gcctggcagcggcaagaaaagagcccactccaccggcaagcaatagctctgagtccctccac 
               
               
                   
               
               
                 ctctcttttggtggggtatccatgtccctcggatcaattttcgacggagagacggcccgcca 
               
               
                   
               
               
                 ggcagcggtacaacccctggcaacaggccccacggatgtgcctatgtctttcggatcgtttt 
               
               
                   
               
               
                 ccgacggagagattgatgagctgagccgcagagtaactgagtccgaacccgtcctgtttgga 
               
               
                   
               
               
                 tcatttgaaccgggcgaagtgaactcaattatatcgtcccgatcagccgtatcttttccact 
               
               
                   
               
               
                 acgcaagcagagacgtagacgcaggagcaggaggactgaatactgactaaccggggtaggtg 
               
               
                   
               
               
                 ggtacatattttcgacggacacaggccctgggcacttgcaaaagaagtccgttctgcagaac 
               
               
                   
               
               
                 cagcttacagaaccgaccttggagcgcaatgtcctggaaagaattcatgccccggtgctcga 
               
               
                   
               
               
                 cacgtcgaaagaggaacaactcaaactcaggtaccagatgatgcccaccgaagccaacaaaa 
               
               
                   
               
               
                 gtaggtaccagtctcgtaaagtagaaaatcagaaagccataaccactgagcgactactgtca 
               
               
                   
               
               
                 ggactacgactgtataactctgccacagatcagccagaatgctataagatcacctatccgaa 
               
               
                   
               
               
                 accattgtactccagtagcgtaccggcgaactactccgatccacagttcgctgtagctgtct 
               
               
                   
               
               
                 gtaacaactatctgcatgagaactatccgacagtagcatcttatcagattactgacgagtac 
               
               
                   
               
               
                 gatgcttacttggatatggtagacgggacagtcgcctgcctggatactgcaaccttctgccc 
               
               
                   
               
               
                 cgctaagcttagaagttacccgaaaaaacatgagtatagagccccgaatatccgcagtgcgg 
               
               
                   
               
               
                 ttccatcagcgatgcagaacacgctacaaaatgtgctcattgccgcaactaaaagaaattgc 
               
               
                   
               
               
                 aacgtcacgcagatgcgtgaactgccaacactggactcagcgacattcaatgtcgaatgctt 
               
               
                   
               
               
                 tcgaaaatatgcatgtaatgacgagtattgggaggagttcgctcggaagccaattaggatta 
               
               
                   
               
               
                 ccactgagtttgtcaccgcatatgtagctagactgaaaggccctaaggccgccgcactattt 
               
               
                   
               
               
                 gcaaagacgtataatttggtcccattgcaagaagtgcctatggatagattcgtcatggacat 
               
               
                   
               
               
                 gaaaagagacgtgaaagttacaccaggcacgaaacacacagaagaaagaccgaaagtacaag 
               
               
                   
               
               
                 tgatacaagccgcagaacccctggcgactgcttacttatgcgggattcaccgggaattagtg 
               
               
                   
               
               
                 cgtaggcttacggccgtcttgcttccaaacattcacacgctttttgacatgtcggcggagga 
               
               
                   
               
               
                 ttttgatgcaatcatagcagaacacttcaagcaaggcgacccggtactggagacggatatcg 
               
               
                   
               
               
                 catcattcgacaaaagccaagacgacgctatggcgttaaccggtctgatgatcttggaggac 
               
               
                   
               
               
                 ctgggtgtggatcaaccactactcgacttgatcgagtgcgcctttggagaaatatcatccac 
               
               
                   
               
               
                 ccatctacctacgggtactcgttttaaattcggggcgatgatgaaatccggaatgttcctca 
               
               
                   
               
               
                 cactttttgtcaacacagttttgaatgtcgttatcgccagcagagtactagaagagcggctt 
               
               
                   
               
               
                 aaaacgtccagatgtgcagcgttcattggcgacgacaacatcatacatggagtagtatctga 
               
               
                   
               
               
                 caaagaaatggctgagaggtgcgccacctggctcaacatggaggttaagatcatcgacgcag 
               
               
                   
               
               
                 tcatcggtgagagaccaccttacttctgcggcggatttatcttgcaagattcggttacttcc 
               
               
                   
               
               
                 acagcgtgccgcgtggcggatcccctgaaaaggctgtttaagttgggtaaaccgctcccagc 
               
               
                   
               
               
                 cgacgacgagcaagacgaagacagaagacgcgctctgctagatgaaacaaaggcgtggttta 
               
               
                   
               
               
                 gagtaggtataacaggcactttagcagtggccgtgacgacccggtatgaggtagacaatatt 
               
               
                   
               
               
                 acacctgtcctactggcattgagaacttttgcccagagcaaaagagcattccaagccatcag 
               
               
                   
               
               
                 aggggaaataaagcatctctacggtggtcctaaatagtcagcatagtacatttcatctgact 
               
               
                   
               
               
                 aatactacaacaccaccacctctagaccatggatcccgtcgttttacaacgtcgtgactggg 
               
               
                   
               
               
                 aaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgt 
               
               
                   
               
               
                 aatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatg 
               
               
                   
               
               
                 gcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttc 
               
               
                   
               
               
                 ctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatc 
               
               
                   
               
               
                 tacaccaacgtaacctatcccattacggtcaatccgccgtttgttcccacggagaatccgac 
               
               
                   
               
               
                 gggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaa 
               
               
                   
               
               
                 ttatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttac 
               
               
                   
               
               
                 ggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaa 
               
               
                   
               
               
                 ccgcctcgcggtgatggtgctgcgttggagtgacggcagttatctggaagatcaggatatgt 
               
               
                   
               
               
                 ggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagc 
               
               
                   
               
               
                 gatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagt 
               
               
                   
               
               
                 tcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaa 
               
               
                   
               
               
                 cgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttat 
               
               
                   
               
               
                 gccgatcgcgtcacactacgtctcaaggtcgaaaacccgaaactgtggagcgccgaaatccc 
               
               
                   
               
               
                 gaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaag 
               
               
                   
               
               
                 cctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaag 
               
               
                   
               
               
                 ccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatgga 
               
               
                   
               
               
                 tgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgct 
               
               
                   
               
               
                 gttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtg 
               
               
                   
               
               
                 gtggatgaagccaatattgaaacccacggcatggtgccaatcaatcgtctgaccgatgatcc 
               
               
                   
               
               
                 gcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacc 
               
               
                   
               
               
                 cgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctg 
               
               
                   
               
               
                 tatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccga 
               
               
                   
               
               
                 caccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcc 
               
               
                   
               
               
                 cggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctg 
               
               
                   
               
               
                 atcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggca 
               
               
                   
               
               
                 ggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgc 
               
               
                   
               
               
                 tgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacg 
               
               
                   
               
               
                 ccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagc 
               
               
                   
               
               
                 gctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcg 
               
               
                   
               
               
                 aagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcg 
               
               
                   
               
               
                 ctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaaca 
               
               
                   
               
               
                 gttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtac 
               
               
                   
               
               
                 gcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcag 
               
               
                   
               
               
                 tggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatct 
               
               
                   
               
               
                 gaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgcc 
               
               
                   
               
               
                 agtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgc 
               
               
                   
               
               
                 gatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattga 
               
               
                   
               
               
                 ccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgt 
               
               
                   
               
               
                 tgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccggtcacgcgtggcag 
               
               
                   
               
               
                 catcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaat 
               
               
                   
               
               
                 ggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctga 
               
               
                   
               
               
                 actgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaac 
               
               
                   
               
               
                 tatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgta 
               
               
                   
               
               
                 taccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatg 
               
               
                   
               
               
                 gcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactg 
               
               
                   
               
               
                 atggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacgg 
               
               
                   
               
               
                 tttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaattcagct 
               
               
                   
               
               
                 gagcgccgttcgctaccattaccagttggtctggtgtcaaaaataataataaccgggcaggg 
               
               
                   
               
               
                 gggatcctagacgctacgccccaatgatccgaccagcaaaactcgatgtacttccgaggaac 
               
               
                   
               
               
                 tgatgtgcataatgcaggaattcgatatcaagctagcatgcaggccttgggcccaatgatcc 
               
               
                   
               
               
                 gaccagcaaaactcgatgtacttccgaggaactgatgtgcataatgcatcaggctggtacat 
               
               
                   
               
               
                 tagatccccgcttaccgcgggcaatatagcaacactaaaaactcgatgtacttccgaggaag 
               
               
                   
               
               
                 cgcagtgcataatgctgcgcagtgttgccacataaccactatattaaccatttatctagcgg 
               
               
                   
               
               
                 acgccaaaaactcaatgtatttctgaggaagcgtggtgcataatgccacgcagcgtctgcat 
               
               
                   
               
               
                 aacttttattatttcttttattaatcaacaaaattttgtttttaacatttcaaaaaaaaaaa 
               
               
                   
               
               
                 aaaaaaaaaaaaaaaaaaaaaaaaaagggaattcctcgattaattaagcggccgc 
               
            
           
         
       
     
     To test cap-independent launch of Sindbis virus replicons, the plasmids pCO390 and Sinrep/lacZ were linearized with Not I and Pac I, respectively, and transcribed in vitro using the SP6 Message Machine kit® (Ambion, Inc., Austin, Tex.). Transcription reactions were performed according to the supplied manual except 10 mM NTP solution (Invitrogen Corp., Carlsbad, Calif.) was substituted for the 2× NTP mix provided in the kit. Following transcription, template DNA was removed by DNase digestion and uncapped RNA was purified using the MEGAClear purification kit® (Ambion, Inc., Austin, Tex.) according to supplied instructions. The resulting uncapped message RNA was introduced into BHK cells by electroporation. 
     For electroporation, late log-phase BHK cells cultured in complete growth medium were trypsinized, washed four times in RNase-free PBS (Ambion, Inc.) and resuspended in RNase-free PBS at 5×10 7  cell/ml. Immediately prior to electroporation, 20 ug RNA in PBS was mixed with 0.5 mL cell suspension and transferred to a chilled electroporation cuvette (0.4 cm gap). The cells were pulsed twice with 1.65 kV, 25 uF capacitance and infinite resistance. Pulsed cells were incubated 10 minutes at room temperature then suspended in 10 mL complete growth medium and plated on a 96-well plate with 100 uL/well. Electroporated cells were incubated overnight at 37° C. then stained for β-galactosidase activity as described previously. 
       FIG. 8B  discloses a study addressed only the issue of the influence of capping on expression from an mRNA encoding β-galactosidase, where the mRNA contained an IRES sequence operably linked with the nucleic acid encoding β-galactosidase ( FIG. 8B ). DI-IRES RNA was electroporated into BHK cells and assayed for lacZ positive cells after 24 hr. 
     The  FIG. 8B  study involved only mRNA electroporated into BHK cells.  Listeria  was not used in the second study, that is,  L. monocytogens  was not used as an intermediary vector. The second study demonstrates expression from the IRES-containing mRNA (capped), and lower expression from the IRES-containing mRNA that is not capped. The figure demonstrates that the viral-derived expression vector (replicon) is functional, even without a cap. The source of the IRES was ECMV (encephalomyocarditis virus). Capped and uncapped RNA was prepared using SP6 Message Machine kit® (Ambion, Inc., Austin, Tex.), where uncapped RNA was made by leaving out the cap analogue from the incubation mixture. Again, the results demonstrate that capping is not a requirement for expression of the DI-IRES RNA. 
     To test the ability of  L. monocytogenes  to deliver Cap-independent replicon RNA to infected eukaryotic cells,  L. monocytogenes  strains are derived that contain a Sindbis virus replicon downstream of an intracellular inducible bacterial promoter. As a non-limiting example, expression cassettes consisting of the actA promoter and Cap-independent Sindbis virus replicons are constructed in the integration vector pINT for stable integration adjacent to the tRNA Arg  locus of the  Listeria  chromosome. The actA promoter is fused to the Cap-independent Sindbis virus replicon from pCO390 by SOE-PCR. The actA promoter including the transcriptional start site are amplified from the plasmid p221 with the primers: 
     
       
         
           
               
            
               
                 BamHI-PactA 
               
            
           
           
               
            
               
                 (SEQ ID NO: 35) 
               
            
           
           
               
            
               
                 5′  GGATCC GGGAAGCAGTTGGGGTTAACTG 3′ 
               
               
                   
               
               
                 PactA DI/IRES REV 
               
            
           
           
               
            
               
                 (SEQ ID NO: 36) 
               
            
           
           
               
            
               
                 5′ CGGGGATACTCACCACTATATCCTTATACTCCCTCCTCGTGATAC 
               
               
                 GC 3′ 
               
            
           
         
       
     
     The 5′ end of the Sindbis virus replicon is amplified from pCO390 with the following primers: 
     
       
         
           
               
            
               
                 PactA DI/IRES FOR 
               
            
           
           
               
            
               
                 (SEQ ID NO: 37) 
               
            
           
           
               
            
               
                 5′ GCGTATCACGAGGAGGGAGTATAAGGATATAGTGGTGAGTATCCC 
               
               
                 CG 3′ 
               
               
                   
               
               
                 WL224 
               
            
           
           
               
            
               
                 (SEQ ID NO: 33) 
               
            
           
           
               
            
               
                 5′ ATAC CGGCCG TGGCTAGTATC 3′ 
               
            
           
         
       
     
     The products of the above amplifications are pooled and used as template in a subsequent amplification with the primer set BamHI-actA/WL224. The resulting 4169 bp product is digested with BamHI and EagI (sites are underlined in respective primers) and cloned into the BamHI-EagI sites of pINT. The remainder of the sinrep replicon, including the lacZ reporter, is isolated from a partial EagI digest of pCO390 and inserted into the EagI site of the pINT intermediate described above. The pINT-derived plasmids are integrated into the chromosome of designated  L. monocytogenes  strains following conjugation with an  E. coli  donor strain and selection on erythromycin. Once integration is confirmed by PCR, pINT vector sequence including the erythromycin resistance marker is excised by transient expression of Cre recombinase. The resulting erythromycin-sensitive strain is then conjugated with an SM10 donor strain containing a second integration vector that inserts at the comK locus of the  L. monocytogenes  chromosome. This second integration vector includes an expression cassette with holin, lysin or holin and lysin downstream of an inducible promoter for intracellular expression of holin or holin and lysin. 
     Additional configurations of the Sindbis virus replicon integrated into the genome of  L. monocytogenes  encoding holin, or holing and lysin to facilitate launch of the Sindbis virus derived replicon RNA in the cytoplasm of infected cells include incorporation of the wild-type 5′end of the Sindbis virus replicon (without the DI tRNA-Asp or ECMV-IRES) or the DI tRNA-Asp alone (without ECMV-IRES) at the 5′end of the replicon. 
     B. Construction of Cap-Independent Poliovirus Replicons. 
     In some embodiments, it is preferred to derive replicons from cap-independent viruses having single-stranded RNA genomes of positive polarity. As a non-limiting example, polioviruses, which belong to the Picornaviridae family, are cap-independent by virtue of an IRES element at the 5′ proximal end of the viral RNA genome, are provided. While replicons derived from polioviruses are described, in can be appreciated by those skilled in the art that such embodiments can be derived from any member of the Picornaviridae family. The poliovirus replicon cDNA was derived by a first RT-PCR step using viral RNA as template that is isolated from a stock of poliovirus Sabin Type 2 human vaccine strain (ATCC VR-301), using the Trizol reagent (Invitrogen, Carlsbad, Calif.). The poliovirus RNA was amplified in two fragments, comprised of a first fragment corresponding to the viral 5′ end to the carboxyl terminus of VP4 (nts. 1-954 according to NCBI accession X00595), and a second fragment corresponding to the amino terminus of the cysteine proteinase 2A viral protein to the viral 3′ end (nts. 3364-7439). Thus, the replicon includes the viral 5′ and 3′ sequences required in cis for replication, the nonstructural proteins which together comprise the viral replicase and proteinase activities for processing of the viral polyprotein, and VP4 as a leader sequence to facilitate efficient translation of a heterologous sequence fused in frame. However, the replicon is deleted of the poliovirus VP2, VP3, and VP1 viral genes and is unable to synthesize productively infectious virus. In some embodiments, the poliovirus replicon was further modified to include a 2A cysteine proteinase recognition amino acid sequence at the junction between the VP4-heterologous antigen fusion protein, as described previously (Porter, et al. (1995) J. Virol. 69:1548-1555). In some embodiments, the poliovirus replicon was still further modified to include two unique restriction endonuclease recognition sites in tandem between the 2A cysteine proteinase recognition amino acid sequence and the amino terminus of the cysteine proteinase 2A viral protein, to facilitate insertion of a sequence encoding a desired protein, as a non-limiting example a tumor antigen or an antigen related to a designated infectious disease, such as hepatitis C virus or influenza. In some embodiments, it is preferred that the sequences of the unique restriction endonuclease recognition sites are configured such that to the translational reading frame of the replicon is maintained following insertion of a desired protein. 
     As a non-limiting example, the poliovirus (PV) replicon cDNA was inserted into the p217 pINT integration vector (patent ref), for insertion into the listerial chromosome, adjacent to the tRNA Arg  gene (Lauer, et al., supra). 
     The first step of the construction was to insert a prokaryotic promoter sequence, as a non-limiting example, the  L. monocytogenes  PrfA-inducible actA core promoter, into the p217 vector. The actA core promoter was amplified with Pfx polymerase (Invitrogen, Carlsbad, Calif.) using the primer set WL289/WL290. The resulting PCR product was purified with the MERmaid kit (Bio 101), digested with Kpn I and Sal I and inserted into the multiple cloning sequence of the p217 pINT integration vector between the Kpn I and Xho I sites. This plasmid is known as pIN548. The +1 transcription initiation sequence of the actA core promoter is shown as a bolded A nucleotide base below in the WL290 reverse primer and corresponds to the authentic 5′ end of the polio virus RNA. 
     Primers for amplification of the actA core promoter sequence with flanking unique 5′ end Kpn I and 3′ Sal I sites (underlined) and “buffer” sequence (lower case) to facilitate restriction enzyme digestion efficiency: 
     
       
         
           
               
            
               
                 WL289: 
               
            
           
           
               
            
               
                 (SEQ ID NO: 38) 
               
            
           
           
               
            
               
                 5′ tatat GGTACC GGGAAGCAGTTGGGGTTAACTG 3′ 
               
               
                   
               
               
                 WL290: 
               
            
           
           
               
            
               
                 (SEQ ID NO: 39) 
               
            
           
           
               
            
               
                 5′ atata GTCGAC   A TTTTAAGAATATCACTTGGAGAATTAATTTTTC 
               
               
                 TC 3′ 
               
            
           
         
       
     
     As a non-limiting example for the derivation of the PV-derived cap-independent replicon cDNA, the first fragment corresponding to the viral 5′ end to the carboxyl terminus of VP4 (nts. 1-954) was generated by RT-PCR using the primers described below: 
     First strand cDNA synthesis: 
     
       
         
           
               
               
            
               
                   
                 WL294 (reverse; viral nts. 1020-999): 
               
            
           
           
               
               
               
            
               
                   
                 5′ CGTTGAATTGCCCAGAGTTAGC 3′ 
                 (SEQ ID NO: 40) 
               
            
           
         
       
     
     First strand cDNA synthesis was accomplished with the AffinityScript First-strand Synthesis System® (Stratagene, San Diego, Calif.) according to the manufacturer&#39;s specifications, using approximately 200 ng of purified PV genomic RNA. The cDNA product was used directly to amplify the 5′ end PV replicon cDNA fragment by a standard PCR protocol, using the primer set shown below: 
     PCR amplification primer set:
 
WL297 (forward; viral nts. 1-23, underlined):
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 41) 
               
            
           
           
               
               
            
               
                   
                 5′ atat-GTCGAC- TTAAAACAGCTCTGGGGTTGTAC  3′ 
               
            
           
         
       
     
     In addition to complementarity with PV nts. 1-23 (underlined), beginning at its 5′ end, primer WL297 contains “buffer” sequence (lowercase) to facilitate restriction endonuclease digestion and a Sal I site. While the ordered elements are shown separated by dashes to facilitate identification of each motif, the primer is synthesized as a single 33 base-long nucleic acid. 
     WL295 [reverse; viral nts. 954-927 (underlined)]: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 42) 
               
            
           
           
               
            
               
                 5′ atta-CGGCCG- 
               
               
                 TCCATATGTGTCGAGCAGTTTTTG- GTTTAGCATGGGAGCGGTCTTAATA   
               
               
                   AGG  3′ 
               
            
           
         
       
     
     In addition to complementarity with PV nts. 954-927 (underlined), beginning at its 5′ end, primer WL295 contains sequences corresponding to the following ordered elements: “buffer” sequence to facilitate restriction endonuclease digestion, Eag I site, and reverse complementary sequence corresponding to the authentic PV 3D polymerase 2A cysteine protease cleavage site (QKLLDTYG, SEQ ID NO:43). While the ordered elements are shown separated by dashes to facilitate identification of each motif, the primer is synthesized as a single 62 base-long nucleic acid. 
     The resulting amplicon product generated from the PCR reaction with the WL297/WL295 primer set was purified over a Qiagen® column, digested with Sal I and Eag I, and inserted into the multiple cloning sequence of the pIN548 integration vector plasmid between the unique Sal I and Eag I sites. This plasmid will be known as pIN586. The sequence of the WL297/WL295 amplicon is shown below. 
     WL297/WL295 amplicon sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 44) 
               
            
           
           
               
            
               
                 ATATGTCGACTTAAAACAGCTCTGGGGTTGTACCCACCCCAGAGGCCCAC 
               
               
                   
               
               
                 GTGGCGGCTAGCACTCCGGTATTACGGTACCCTTGTGCGCCTGTTTTATA 
               
               
                   
               
               
                 CTCCCCTCCCGCAACTTAGAAGCACGAAACCAAGTTCAATAGAAGGGGGT 
               
               
                   
               
               
                 ACAAACCAGTACCACTACGAACAAGCACTTCTGTTTCCCCGGTGACATTG 
               
               
                   
               
               
                 CATAGACTGCTCACGCGGTTGAAAGTGATCGATCCGTTACCCGCTTGTGT 
               
               
                   
               
               
                 ACTTCGAAAAGCCTAGTATCGCCTTGGAATCTTCGACGCGTTGCGCTCAG 
               
               
                   
               
               
                 CACCCGACCCCGGGGTGTAGCTTAGGCTGATGAGTCTGGACATTCCTCAC 
               
               
                   
               
               
                 CGGTGACGGTGGTCCAGGCTGCGTTGGCGGCCTACCTATGGCTAACGCCA 
               
               
                   
               
               
                 TAGGACGTTAGATGTGAACAAGGTGTGAAGAGCCTATTGAGCTACATAAG 
               
               
                   
               
               
                 AGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACCACGGAACAGGCGGTC 
               
               
                   
               
               
                 GCGAACCAGTGACTGGCTTGTCGTAACGCGCAAGTCTGTGGCGGAACCGA 
               
               
                   
               
               
                 CTACTTTGGGTGTCCGTGTTTCCTGTTATTTTTATCATGGCTGCTTATGG 
               
               
                   
               
               
                 TGACAATCAGAGATTGTTATCATAAAGCGAATTGGATTGGCCATCCGGTG 
               
               
                   
               
               
                 AGTGTTGTGTCAGGTATACAACTGTTTGTTGGAACCACTGTGTTAGCTTT 
               
               
                   
               
               
                 ACTTCTCATTTAACCAATTAATCAAAAACAATACGAGGATAAAACAACAA 
               
               
                   
               
               
                 TACTACAATGGGCGCCCAAGTTTCATCACAGAAAGTTGGAGCCCACGAAA 
               
               
                   
               
               
                 ATTCAAACAGAGCCTATGGCGGGTCCACCATCAATTACACTACAATCAAT 
               
               
                   
               
               
                 TACTATAGGGACTCTGCAAGCAATGCAGCAAGCAAGCAAGATTTTGCACA 
               
               
                   
               
               
                 AGATCCGTCCAAGTTCACCGAACCCATTAAGGACGTCCTTATTAAGACCG 
               
               
                   
               
               
                 CTCCCATGCTAAACCAAAAACTGCTCGACACATATGGACGGCCGTAAT 
               
            
           
         
       
     
     As a non-limiting example for the derivation of the poliovirus-derived cap-independent replicon cDNA, the second fragment corresponding to the amino terminus of the cysteine proteinase 2A viral protein to the viral 3′ end (nts. 3386-7440) was generated by RT-PCR using the primers described below: 
     First strand cDNA synthesis:
 
WL282 (reverse; viral nts. 5067-5055, underlined):
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 45) 
               
            
           
           
               
            
               
                 5′ TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT- CTCCGA   
               
               
                   ATTAAAG  3′ 
               
            
           
         
       
     
     In addition to complementarity with PV nts. 7439-7427 (underlined), beginning at its 5′ end, primer WL282 also preferably contains 40 consecutive T residues that are complementary with the PV 3′ end polyadenylation sequence, or poly(A) tail. While the ordered elements are shown separated by dashes to facilitate identification of each motif, the primer is synthesized as a single 53 base-long nucleic acid. 
     First strand cDNA synthesis was accomplished with the AffinityScript First-strand Synthesis System® (Stratagene, San Diego, Calif.) according to the manufacturer&#39;s specifications, using approximately 200 ng of purified PV genomic RNA. The cDNA product was used directly to amplify the 5′ end PV replicon cDNA fragment by a standard PCR protocol, using the primer set shown below: 
     PCR amplification primer set:
 
WL296 (forward; viral nts. 3364-3392, underlined):
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 46) 
               
            
           
           
               
            
               
                 5′ atta-CGGCCGTTTAAACCCTGCAGG- GAAAAGGGATTAACGACTTA   
               
               
                   TGGATTTGG  3′ 
               
            
           
         
       
     
     In addition to PV nts. 3364-3392 (underlined), beginning at its 5′ end, primer WL296 contains sequences corresponding to the following ordered elements: buffer sequence to facilitate endonuclease digestion, overlapping EagI and PmeI sites and an Sbf I site. While the ordered elements are shown separated by dashes to facilitate identification of each motif, the primer is synthesized as a single 54 base-long nucleic acid. 
     WL298 (reverse; viral nts. 5067-5055, underlined): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 47) 
               
            
           
           
               
            
               
                 5′ gcgc-TTAATTAA-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 
               
               
                 TTTTTTT- CTCCGAATTAAAG  3′ 
               
            
           
         
       
     
     In addition to complementarity with PV nts. 7439-7427 (underlined), beginning at its 5′ end, primer WL298 also preferably contains buffer sequence to facilitate endonuclease digestion (lower case), a Pac I site, followed by 40 consecutive T residues that are complementary with the PV 3′ end polyadenylation sequence. While the ordered elements are shown separated by dashes to facilitate identification of each motif, the primer is synthesized as a single 65 base-long nucleic acid. 
     The resulting amplicon product generated from the PCR reaction with the WL296/WL298 primer set was purified over a Qiagen® column, digested with Eag I and Pac I, and then cloned into the the multiple cloning sequence of the pIN586 integration vector plasmid between the unique EagI and Pad sites. This plasmid is known as pIN599. The sequence of the WL296/WL298 amplicon is shown below. 
     WL296/WL298 amplicon sequence: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 48) 
                   
               
            
           
           
               
               
            
               
                 ATTACGGCCGTTTAAACCCTGCAGGGAAAAGGGATTAACGACTTATGGATTTGGACACCAAA 
                   
               
               
                   
               
               
                 ACAAAGCTGTGTACACAGCTGGCTACAAAATTTGCAATTACCACCTAGCTACACAAGAAGAC 
               
               
                   
               
               
                 TTGCAAAATGCCGTGAGTGTCATGTGGAACAGAGACCTCTTAGTGGCTGAATCAAGGGCCCT 
               
               
                   
               
               
                 TGGCACCGACTCGATCGCAAGGTGCAGCTGTAACACGGGTGTGTACTACTGTGAATCCAGGA 
               
               
                   
               
               
                 GAAAATATTATCCAGTTTCTTTCATTGGGCCCACCTTCCAATACATGGAAGCCAATGAATAT 
               
               
                   
               
               
                 TACCCGGCTAGATATCAATCACACATGCTTATTGGTCATGGGTTTGCATCACCGGGTGATTG 
               
               
                   
               
               
                 TGGTGGCATACTTAGATGTCAACACGGGGTGATAGGAATAATCACTGCTGGTGGGGAAGGCT 
               
               
                   
               
               
                 TGGTTGCATTTTCAGACATTAGAGACCTGTATGCTTATGAGGAGGAAGCTATGGAGCAGGGC 
               
               
                   
               
               
                 ATTTCCAACTATATTGAGTCACTTGGTGCTGCATTTGGTAGTGGATTCACTCAACAAATTGG 
               
               
                   
               
               
                 TGATAAAGTTTCCGAGCTAACCAGCATGGTAACTAGCACCATTACAGAGAAGTTGCTTAAAA 
               
               
                   
               
               
                 ACTTAATCAAAATTATCTCATCACTTGTGATCATTACCAGGAATTATGAGGACACTACCACA 
               
               
                   
               
               
                 GTGCTTGCCACCCTCGCCCTCCTTGGGTGCGACATCTCACCGTGGCAGTGGCTAAAGAAGAA 
               
               
                   
               
               
                 GGCATGTGACATCCTGGAAATTCCATACGCCATCAAACAAGGAGATAGTTGGTTGAAGAAAT 
               
               
                   
               
               
                 TCACTGAGGCATGTAATGCTGCAAAGGGACTGGAGTGGGTGTCCAATAAGATATCCAAATTC 
               
               
                   
               
               
                 ATTAGTTGGTTGCAGGATAAAATCATCCCACAAGCGAGAGACAAATTAGAGTTTGTCACTAA 
               
               
                   
               
               
                 ACTAAAGCAATTAGAAATGCTTGAAAATCAGATTTCCACCATACACCAATCTTGTCCAAGTC 
               
               
                   
               
               
                 AAGAACATCAGGAGATCTTATTCAACAATGTGCGGTGGCTATCTATCCAGTCCAAGAGGTTT 
               
               
                   
               
               
                 GCACCACTATATGCACATGAAGCTAAAAGGATTCAAAAGCTGGAGCATACCATAAATAATTA 
               
               
                   
               
               
                 CGTACAGTTCAAGAGCAAGCACCGTATTGAGCCAGTATGTTTGTTAGTACATGGCAGTCCAG 
               
               
                   
               
               
                 GGACAGGAAAATCAGTTGCAACCAATCTAATTGCTAGAGCAATAGCCGAGAAAGAGAACACC 
               
               
                   
               
               
                 TCCACATACTCACTGCCACCTGATCCGTCTCACTTTGATGGCTACAAGCAACAGGGTGTGGT 
               
               
                   
               
               
                 TATTATGGATGACCTAAACCAAAATCCAGACGGAGCAGACATGAAACTTTTTTGTCAAATGG 
               
               
                   
               
               
                 TGTCCACTGTGGAGTTTATTCCACCGATGGCCTCGCTAGAAGAGAAAGGCATTTTGTTCACA 
               
               
                   
               
               
                 TCTAATTACGTTTTAGCCTCCACCAACTCCAGTCGGATCACACCACCCACGGTGGCTCACAG 
               
               
                   
               
               
                 TGATGCGCTGGCCAGGAGATTCGCATTTGACGTGGACATACAAGTCATGAGCGAGTACTCCA 
               
               
                   
               
               
                 GAGACGGAAAGCTCAACATGGCAATGGCTACTGAAATGTGCAAAAACTGTCATCAACCAGCA 
               
               
                   
               
               
                 AACTTCAAAAGATGTTGTCCTTTAGTGTGTGGCAAGGCAATTCAGTTAATGGATAAATCTTC 
               
               
                   
               
               
                 CAGGGTTAGATACAGCATTGATCAGATCACTACAATGATTGTTAATGAGAGAAACAGAAGAT 
               
               
                   
               
               
                 CAAACATTGGTAATTGCATGGAAGCTCTATTCCAGGGACCACTGCAGTATAAAGATCTAAAA 
               
               
                   
               
               
                 ATAGATGTTAAGACCAGTCCCCCTCCGGAGTGTATCAACGATTTGCTCCAGGCAGTTGATTC 
               
               
                   
               
               
                 CCAGGAAGTGAGAGATTACTGTGAAAAGAAAGGCTGGATTGTTAACATTACCAGTCAGGTTC 
               
               
                   
               
               
                 AAACAGAGAGGAACATCAACCGGGCGATGACTATCCTACAAGCAGTAACTACTTTCGCTGCA 
               
               
                   
               
               
                 GTAGCCGGTGTCGTGTACGTTATGTACAAGCTGTTCGCTGGGCACCAGGGTGCATACACTGG 
               
               
                   
               
               
                 TTTGCCAAATAAACGACCCAATGTACCCACTATCAGGACAGCAAAAGTGCAAGGCCCTGGGT 
               
               
                   
               
               
                 TTGATTACGCAGTGGCCATGGCTAAAAGAAACATTGTTACAGCAACCACCAGCAAAGGGGAG 
               
               
                   
               
               
                 TTTACGATGTTGGGAGTCTATGATAATGTGGCCATCTTGCCAACCCACGCCTCACCTGGTGA 
               
               
                   
               
               
                 AAGCATTGCGATCGACGGTAAAGAGGTGGAAATTCTTGACGCCAAAGCCCTTGAAGATCAGG 
               
               
                   
               
               
                 CAGGAACTAATCTTGAAATTACCATAATTACACTAAAGAGGAACGAGAAGTTCAGAGATATC 
               
               
                   
               
               
                 AGGCCACACATTCCCACCCAAATCACCGAAACAAATGATGGAGTTTTGATCGTGAACACTAG 
               
               
                   
               
               
                 TAAGTACCCCAACATGTATGTTCCCGTTGGTGCTGTGACCGAACAGGGGTATCTTAATCTCG 
               
               
                   
               
               
                 GTGGACGACAAACCGCTCGTACGCTAATGTACAACTTTCCAACTAGAGCAGGTCAGTGTGGT 
               
               
                   
               
               
                 GGTGTCATCACGTGCACTGGTAAAGTCATTGGGATGCATGTTGGTGGGAACGGTTCACATGG 
               
               
                   
               
               
                 GTTCGCGGCGGCCCTAAAGCGGTCATACTTCACTCAGATTCAAGGTGAGATTCAATGGATGA 
               
               
                   
               
               
                 AACCATCAAAAGAAGTGGGATACCCGATCATAAATGCTCCGTCCAAAACCAAACTTGAACCC 
               
               
                   
               
               
                 AGCGCTTTTCACTATGTGTTTGAAGGGGTGAAGGAACCAGCAGTCCTTACCAAAAATGATCC 
               
               
                   
               
               
                 CAGGCTCAGGACAGACTTTGAAGAAGCAATATTCTCTAAGTATGTAGGCAACAAGATCACTG 
               
               
                   
               
               
                 ATGTGGATGAGTACATGAAAGAGGCAGTGGATCATTACGCTGGCCAACTCATGTCTCTAGAC 
               
               
                   
               
               
                 ATCAACACAGAACAAATGTGCTTGGAGGACGCCATGTACGGCACCGATGGCCTGGAAGCACT 
               
               
                   
               
               
                 TGACTTGACCACTAGTGCTGGATACCCTTATGTAGCAATGGGAAAGAAAAAGAGAGACATCT 
               
               
                   
               
               
                 TGAATAAGCAGACTAGAGACACCAAGGAAATGCGGAGACTCTTAGATACTTATGGAATTAAC 
               
               
                   
               
               
                 TTACCGCTTGTAACATATGTTAAAGATGAACTAAGGTCAAAAACTAAGGTGGAGCAGGGAAA 
               
               
                   
               
               
                 ATCCAGATTGATTGAAGCCTCCAGTTTGAATGATTCAGTGGCCATGAGAATGGCATTTGGAA 
               
               
                   
               
               
                 ATCTCTATGCAGCATTTCACAAAAACCCAGGAGTTGTCACTGGCAGTGCAGTTGGTTGTGAT 
               
               
                   
               
               
                 CCAGATCTATTTTGGAGCAAGATCCCAGTGCTAATGGAAGAGAAGCTCTTTGCTTTTGACTA 
               
               
                   
               
               
                 CACAGGTTATGATGCATCACTCAGCCCGGCCTGGTTTGAGGCACTCAAAATGGTGCTAGAGA 
               
               
                   
               
               
                 AAATCGGATTTGGGGACAGGGTGGATTATATTGATTACCTCAACCATTCCCACCACCTGTAC 
               
               
                   
               
               
                 AAAAACAAAACTTATTGCGTAAAAGGCGGCATGCCATCTGGCTGCTCAGGCACATCAATTTT 
               
               
                   
               
               
                 TAACTCAATGATTAACAACTTAATCATTAGGACACTCCTACTGAAAACCTACAAGGGCATAG 
               
               
                   
               
               
                 ATTTAGATCACCTAAAGATGATTGCCTATGGTGATGATGTAATTGCTTCCTACCCCCATGAG 
               
               
                   
               
               
                 GTTGATGCTAGTCTCCTAGCCCAATCAGGAAAAGACTATGGACTAACCATGACTCCAGCAGA 
               
               
                   
               
               
                 CAAGTCAGCTACCTTTGAAACAGTCACATGGGAGAATGTAACATTCTTGAAAAGATTCTTTA 
               
               
                   
               
               
                 GAGCGGATGAGAAGTATCCCTTCCTCATACATCCAGTAATGCCAATGAAGGAGATTCATGAA 
               
               
                   
               
               
                 TCAATTAGATGGACAAAGGATCCCAGAAACACACAGGATCACGTGCGCTCATTGTGCCTATT 
               
               
                   
               
               
                 GGCCTGGCACAACGGCGAAGAAGAATACAACAAGTTCTTAGCTAAAATCAGGAGTGTGCCAA 
               
               
                   
               
               
                 TTGGGAGAGCTTTATTGCTCCCAGAGTACTCTACATTGTACCGCCGTTGGCTCGACTCTTTT 
               
               
                   
               
               
                 TAGTAACCCTACCTCAGTCGAATTGGATTGGGTCATGCTGTTGTAGGGGTAAATTTTTCTTT 
               
               
                   
               
               
                 AATTCGGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTAATTAAGCGC 
               
            
           
         
       
     
     The final step in the construction of the PV replicon cDNA was to functionally link the actA promoter core sequence with the authentic 5′ end of the PV replicon cDNA by splice overlap extension (SOE) PCR. The SOE PCR step removed the Sal I GTCGAC recognition site intervening the actA promoter core and the PV 5′ end of the pIN599 plasmid. Upon deletion of the Sal I site, this plasmid was designated as pIN662. The DNA sequence corresponding to the actA core promoter and polio replicon from pIN662 is shown below. 
     actA-Pcore-PVrep sequence from pIN662: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 49) 
                   
               
            
           
           
               
               
            
               
                 CGGGAAGCAGTTGGGGTTAACTGATTAACAAATGTTAGAGAAAAATTAATTCTCCAAGTGAT 
                   
               
               
                   
               
               
                 ATTCTTAAAATTTAAAACAGCTCTGGGGTTGTACCCACCCCAGAGGCCCACGTGGCGGCTAG 
               
               
                   
               
               
                 CACTCCGGTATTACGGTACCCTTGTGCGCCTGTTTTATACTCCCCTCCCGCAACTTAGAAGC 
               
               
                   
               
               
                 ACGAAACCAAGTTCAATAGAAGGGGGTACAAACCAGTACCACTACGAACAAGCACTTCTGTT 
               
               
                   
               
               
                 TCCCCGGTGACATTGCATAGACTGCTCACGCGGTTGAAAGTGATCGATCCGTTACCCGCTTG 
               
               
                   
               
               
                 TGTACTTCGAAAAGCCTAGTATCGCCTTGGAATCTTCGACGCGTTGCGCTCAGCACCCGACC 
               
               
                   
               
               
                 CCGGGGTGTAGCTTAGGCTGATGAGTCTGGACATTCCTCACCGGTGACGGTGGTCCAGGCTG 
               
               
                   
               
               
                 CGTTGGCGGCCTACCTATGGCTAACGCCATAGGACGTTAGATGTGAACAAGGTGTGAAGAGC 
               
               
                   
               
               
                 CTATTGAGCTACATAAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACCACGGAACAGG 
               
               
                   
               
               
                 CGGTCGCGAACCAGTGACTGGCTTGTCGTAACGCGCAAGTCTGTGGCGGAACCGACTACTTT 
               
               
                   
               
               
                 GGGTGTCCGTGTTTCCTGTTATTTTTATCATGGCTGCTTATGGTGACAATCAGAGATTGTTA 
               
               
                   
               
               
                 TCATAAAGCGAATTGGATTGGCCATCCGGTGAGTGTTGTGTCAGGTATACAACTGTTTGTTG 
               
               
                   
               
               
                 GAACCACTGTGTTAGCTTTACTTCTCATTTAACCAATTAATCAAAAACAATACGAGGATAAA 
               
               
                   
               
               
                 ACAACAATACTACAATGGGCGCCCAAGTTTCATCACAGAAAGTTGGAGCCCACGAAAATTCA 
               
               
                   
               
               
                 AACAGAGCCTATGGCGGGTCCACCATCAATTACACTACAATCAATTACTATAGGGACTCTGC 
               
               
                   
               
               
                 AAGCAATGCAGCAAGCAAGCAAGATTTTGCACAAGATCCGTCCAAGTTCACCGAACCCATTA 
               
               
                   
               
               
                 AGGACGTCCTTATTAAGACCGCTCCCATGCTAAACCAAAAACTGCTCGACACATATGGACGG 
               
               
                   
               
               
                 CCGTTTAAACCCTGCAGGGAAAAGGGATTAACGACTTATGGATTTGGACACCAAAACAAAGC 
               
               
                   
               
               
                 TGTGTACACAGCTGGCTACAAAATTTGCAATTACCACCTAGCTACACAAGAAGACTTGCAAA 
               
               
                   
               
               
                 ATGCCGTGAGTGTCATGTGGAACAGAGACCTCTTAGTGGCTGAATCAAGGGCCCTTGGCACC 
               
               
                   
               
               
                 GACTCGATCGCAAGGTGCAGCTGTAACACGGGTGTGTACTACTGTGAATCCAGGAGAAAATA 
               
               
                   
               
               
                 TTATCCAGTTTCTTTCATTGGGCCCACCTTCCAATACATGGAAGCCAATGAATATTACCCGG 
               
               
                   
               
               
                 CTAGATATCAATCACACATGCTTATTGGTCATGGGTTTGCATCACCGGGTGATTGTGGTGGC 
               
               
                   
               
               
                 ATACTTAGATGTCAACACGGGGTGATAGGAATAATCACTGCTGGTGGGGAAGGCTTGGTTGC 
               
               
                   
               
               
                 ATTTTCAGACATTAGAGACCTGTATGCTTATGAGGAGGAAGCTATGGAGCAGGGCATTTCCA 
               
               
                   
               
               
                 ACTATATTGAGTCACTTGGTGCTGCATTTGGTAGTGGATTCACTCAACAAATTGGTGATAAA 
               
               
                   
               
               
                 GTTTCCGAGCTAACCAGCATGGTAACTAGCACCATTACAGAGAAGTTGCTTAAAAACTTAAT 
               
               
                   
               
               
                 CAAAATTATCTCATCACTTGTGATCATTACCAGGAATTATGAGGACACTACCACAGTGCTTG 
               
               
                   
               
               
                 CCACCCTCGCCCTCCTTGGGTGCGACATCTCACCGTGGCAGTGGCTAAAGAAGAAGGCATGT 
               
               
                   
               
               
                 GACATCCTGGAAATTCCATACGCCATCAAACAAGGAGATAGTTGGTTGAAGAAATTCACTGA 
               
               
                   
               
               
                 GGCATGTAATGCTGCAAAGGGACTGGAGTGGGTGTCCAATAAGATATCCAAATTCATTAGTT 
               
               
                   
               
               
                 GGTTGCAGGATAAAATCATCCCACAAGCGAGAGACAAATTAGAGTTTGTCACTAAACTAAAG 
               
               
                   
               
               
                 CAATTAGAAATGCTTGAAAATCAGATTTCCACCATACACCAATCTTGTCCAAGTCAAGAACA 
               
               
                   
               
               
                 TCAGGAGATCTTATTCAACAATGTGCGGTGGCTATCTATCCAGTCCAAGAGGTTTGCACCAC 
               
               
                   
               
               
                 TATATGCACATGAAGCTAAAAGGATTCAAAAGCTGGAGCATACCATAAATAATTACGTACAG 
               
               
                   
               
               
                 TTCAAGAGCAAGCACCGTATTGAGCCAGTATGTTTGTTAGTACATGGCAGTCCAGGGACAGG 
               
               
                   
               
               
                 AAAATCAGTTGCAACCAATCTAATTGCTAGAGCAATAGCCGAGAAAGAGAACACCTCCACAT 
               
               
                   
               
               
                 ACTCACTGCCACCTGATCCGTCTCACTTTGATGGCTACAAGCAACAGGGTGTGGTTATTATG 
               
               
                   
               
               
                 GATGACCTAAACCAAAATCCAGACGGAGCAGACATGAAACTTTTTTGTCAAATGGTGTCCAC 
               
               
                   
               
               
                 TGTGGAGTTTATTCCACCGATGGCCTCGCTAGAAGAGAAAGGCATTTTGTTCACATCTAATT 
               
               
                   
               
               
                 ACGTTTTAGCCTCCACCAACTCCAGTCGGATCACACCACCCACGGTGGCTCACAGTGATGCG 
               
               
                   
               
               
                 CTGGCCAGGAGATTCGCATTTGACGTGGACATACAAGTCATGAGCGAGTACTCCAGAGACGG 
               
               
                   
               
               
                 AAAGCTCAACATGGCAATGGCTACTGAAATGTGCAAAAACTGTCATCAACCAGCAAACTTCA 
               
               
                   
               
               
                 AAAGATGTTGTCCTTTAGTGTGTGGCAAGGCAATTCAGTTAATGGATAAATCTTCCAGGGTT 
               
               
                   
               
               
                 AGATACAGCATTGATCAGATCACTACAATGATTGTTAATGAGAGAAACAGAAGATCAAACAT 
               
               
                   
               
               
                 TGGTAATTGCATGGAAGCTCTATTCCAGGGACCACTGCAGTATAAAGATCTAAAAATAGATG 
               
               
                   
               
               
                 TTAAGACCAGTCCCCCTCCGGAGTGTATCAACGATTTGCTCCAGGCAGTTGATTCCCAGGAA 
               
               
                   
               
               
                 GTGAGAGATTACTGTGAAAAGAAAGGCTGGATTGTTAACATTACCAGTCAGGTTCAAACAGA 
               
               
                   
               
               
                 GAGGAACATCAACCGGGCGATGACTATCCTACAAGCAGTAACTACTTTCGCTGCAGTAGCCG 
               
               
                   
               
               
                 GTGTCGTGTACGTTATGTACAAGCTGTTCGCTGGGCACCAGGGTGCATACACTGGTTTGCCA 
               
               
                   
               
               
                 AATAAACGACCCAATGTACCCACTATCAGGACAGCAAAAGTGCAAGGCCCTGGGTTTGATTA 
               
               
                   
               
               
                 CGCAGTGGCCATGGCTAAAAGAAACATTGTTACAGCAACCACCAGCAAAGGGGAGTTTACGA 
               
               
                   
               
               
                 TGTTGGGAGTCTATGATAATGTGGCCATCTTGCCAACCCACGCCTCACCTGGTGAAAGCATT 
               
               
                   
               
               
                 GCGATCGACGGTAAAGAGGTGGAAATTCTTGACGCCAAAGCCCTTGAAGATCAGGCAGGAAC 
               
               
                   
               
               
                 TAATCTTGAAATTACCATAATTACACTAAAGAGGAACGAGAAGTTCAGAGATATCAGGCCAC 
               
               
                   
               
               
                 ACATTCCCACCCAAATCACCGAAACAAATGATGGAGTTTTGATCGTGAACACTAGTAAGTAC 
               
               
                   
               
               
                 CCCAACATGTATGTTCCCGTTGGTGCTGTGACCGAACAGGGGTATCTTAATCTCGGTGGACG 
               
               
                   
               
               
                 ACAAACCGCTCGTACGCTAATGTACAACTTTCCAACTAGAGCAGGTCAGTGTGGTGGTGTCA 
               
               
                   
               
               
                 TCACGTGCACTGGTAAAGTCATTGGGATGCATGTTGGTGGGAACGGTTCACATGGGTTCGCG 
               
               
                   
               
               
                 GCGGCCCTAAAGCGGTCATACTTCACTCAGATTCAAGGTGAGATTCAATGGATGAAACCATC 
               
               
                   
               
               
                 AAAAGAAGTGGGATACCCGATCATAAATGCTCCGTCCAAAACCAAACTTGAACCCAGCGCTT 
               
               
                   
               
               
                 TTCACTATGTGTTTGAAGGGGTGAAGGAACCAGCAGTCCTTACCAAAAATGATCCCAGGCTC 
               
               
                   
               
               
                 AGGACAGACTTTGAAGAAGCAATATTCTCTAAGTATGTAGGCAACAAGATCACTGATGTGGA 
               
               
                   
               
               
                 TGAGTACATGAAAGAGGCAGTGGATCATTACGCTGGCCAACTCATGTCTCTAGACATCAACA 
               
               
                   
               
               
                 CAGAACAAATGTGCTTGGAGGACGCCATGTACGGCACCGATGGCCTGGAAGCACTTGACTTG 
               
               
                   
               
               
                 ACCACTAGTGCTGGATACCCTTATGTAGCAATGGGAAAGAAAAAGAGAGACATCTTGAATAA 
               
               
                   
               
               
                 GCAGACTAGAGACACCAAGGAAATGCGGAGACTCTTAGATACTTATGGAATTAACTTACCGC 
               
               
                   
               
               
                 TTGTAACATATGTTAAAGATGAACTAAGGTCAAAAACTAAGGTGGAGCAGGGAAAATCCAGA 
               
               
                   
               
               
                 TTGATTGAAGCCTCCAGTTTGAATGATTCAGTGGCCATGAGAATGGCATTTGGAAATCTCTA 
               
               
                   
               
               
                 TGCAGCATTTCACAAAAACCCAGGAGTTGTCACTGGCAGTGCAGTTGGTTGTGATCCAGATC 
               
               
                   
               
               
                 TATTTTGGAGCAAGATCCCAGTGCTAATGGAAGAGAAGCTCTTTGCTTTTGACTACACAGGT 
               
               
                   
               
               
                 TATGATGCATCACTCAGCCCGGCCTGGTTTGAGGCACTCAAAATGGTGCTAGAGAAAATCGG 
               
               
                   
               
               
                 ATTTGGGGACAGGGTGGATTATATTGATTACCTCAACCATTCCCACCACCTGTACAAAAACA 
               
               
                   
               
               
                 AAACTTATTGCGTAAAAGGCGGCATGCCATCTGGCTGCTCAGGCACATCAATTTTTAACTCA 
               
               
                   
               
               
                 ATGATTAACAACTTAATCATTAGGACACTCCTACTGAAAACCTACAAGGGCATAGATTTAGA 
               
               
                   
               
               
                 TCACCTAAAGATGATTGCCTATGGTGATGATGTAATTGCTTCCTACCCCCATGAGGTTGATG 
               
               
                   
               
               
                 CTAGTCTCCTAGCCCAATCAGGAAAAGACTATGGACTAACCATGACTCCAGCAGACAAGTCA 
               
               
                   
               
               
                 GCTACCTTTGAAACAGTCACATGGGAGAATGTAACATTCTTGAAAAGATTCTTTAGAGCGGA 
               
               
                   
               
               
                 TGAGAAGTATCCCTTCCTCATACATCCAGTAATGCCAATGAAGGAGATTCATGAATCAATTA 
               
               
                   
               
               
                 GATGGACAAAGGATCCCAGAAACACACAGGATCACGTGCGCTCATTGTGCCTATTGGCCTGG 
               
               
                   
               
               
                 CACAACGGCGAAGAAGAATACAACAAGTTCTTAGCTAAAATCAGGAGTGTGCCAATTGGGAG 
               
               
                   
               
               
                 AGCTTTATTGCTCCCAGAGTACTCTACATTGTACCGCCGTTGGCTCGACTCTTTTTAGTAAC 
               
               
                   
               
               
                 CCTACCTCAGTCGAATTGGATTGGGTCATGCTGTTGTAGGGGTAAATTTTTCTTTAATTCGG 
               
               
                   
               
               
                 AGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTAATTAA 
               
            
           
         
       
     
     A heterologous sequence, encoding as non-limiting examples a desired tumor antigen(s) (e.g., Mesothelin), infectious disease antigens including as non-limiting examples hepatitis C virus (e.g., core, NS3, NS5b), human immunodeficiency virus (e.g., gag, env, pol, nef), influenza virus (e.g., hemagglutinin (HA), neuraminidase (NA)), or biochemical reporters (e.g. β-galactosidase), can be inserted between unique Eag I, Pme I and Sbf I sites present between the VP4 and 2A cysteine proteinase encoding sequences of the pIN662 plasmid. In this configuration, the antigen is cleaved from the PV replicon expressed polyprotein by the autocatalytic processing activity of the PV 2A cysteine proteinase. As a non-limiting example, a fusion protein consisting of an ovalbumin epitope SL8 and b-galactosidase was inserted into the EagI site of pIN662 to construct the plasmid pBHE893. 
     Following insertion of the heterologous sequences into the pIN662 plasmid and confirming the fidelity of the PV replicon cDNA by sequence analysis, the resultling plasmid was integrated at the tRNA Arg  locus in the genome of selected  L. monocytogenes  strains using previously described methods (Lauer, et al. (2002) J. Bacteriol. 184:4177-4186). Integration was confirmed on chloramphenicol resistant  L. monocytogenes  colonies by PCR with NC16 (5′ GTCAAAACATACGCTCTTATC 3′; SEQ ID NO:50) and PL95 (5′ ACATAATCAGTCCAAAGTAGATGC 3′; SEQ ID NO:51). 
     In order to test the ability of  L. monocytogenes  to deliver a functional polio virus derived (PV) replicon RNA to infected cells, the plasmid pBHE893 was integrated into selected strains of  L. monocytogenes,  including strains expressing holin and lysin, and the resulting strains were then used to infect BHK cells as described previously. Briefly, BHK cells were seeded at 2×10 4  cells per well in a white 96-well tissue culture plate with a clear bottom (Optilux, BD Biosciences, Franklin Lake, N.J.). Cells were cultured overnight in complete growth medium without antibiotics. BHK cells were infected at a multiplicity of 200 cfu per cell with fresh overnight cultures of  Listeria  strains grown at 30° C. in BHI broth. Strains used in this study are listed in Table 9. Infected cells were cultured for 24 hours at 37° C. in complete growth medium containing 50 μg/mL gentamycin to inhibit extracellular growth of  Listeria.  At 24 hours post-infection, cells were fixed in formaldehyde and stained for β-galactosidase activity as described previously ( FIG. 9 ). Infected cells were imaged and cells positive for b-galactosidase were enumerated using an ImmunoSpot plate reader (CTL, Cleveland, Ohio).  FIG. 9  shows that β-galactosidase activity in BHK cells was dependent on infection with  L. monocytogenes  strains that carry nucleic acids encoding viral replicons. The number of cells positive for β-galactosidase activity was greatly enhanced upon infection with  L. monocytogenes  strains harboring a PV and also co-expressing both holin and lysin, indicating that  L. monocytogenes  can deliver nucleic acids to infected cells and that the lytic activity of holin and lysin provide a means by which functional replicon nucleic acids can be released into the cytosol of infected cells ( FIG. 9 ). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Lm strains for delivering cap-independent viral-based replicon 
               
               
                 to the cytoplasm of a mammalian host cell. 
               
            
           
           
               
               
               
               
            
               
                 Strain 
                 Background 
                 Holin/lysin 
                 Replicon 
               
               
                   
               
               
                 DP-L4056 
                 WT 
                 − 
                 none 
               
               
                 Lm277 
                 WT 
                 + (holin only) 
                 Sindbis virus (DNA delivery) 
               
               
                 BH1151 
                 WT 
                 − 
                 Polio (RNA delivery) 
               
               
                 BH1145 
                 WT 
                 + 
                 Polio (RNA delivery) 
               
               
                 BH1150 
                 ΔactAΔinlB 
                 − 
                 Polio (RNA delivery) 
               
               
                 BH1147 
                 ΔactAΔinlB 
                 + 
                 Polio (RNA delivery) 
               
               
                   
               
            
           
         
       
     
     In another embodiment, the PV replicon decribed above was cloned downstream of the full-length actA promoter from  L. monocytogenes.  In addition to the core sequence sufficient for intracellular inducible expression, the full length actA promoter includes the 5′ untranslated region of the actA transcript, 150 nt in length, that results in enhanced actA expression in the cytoplasm of infected host cells. The PV replicon was cloned downstream of the full length actA promoter to test the transcriptional activity of the full-length actA promoter. The full-length actA promoter was amplified from  L. monocytogenes  genomic DNA and spliced to the 5′end of the polio replicon in by SOE-PCR. The DNA sequence corresponding to the full-length actA promoter is shown below. 
                    (SEQ ID NO: 52)                 gggaagcagttggggttaactgattaacaaatgttagagaaaaattaatt               ctccaagtgatattcttaaaa T aattcatgaatattttttcttatattag               actattaagaagataattaactgctaatccaatttttaacggaataaatt               agtgaaaatgaaggccgaattttccttgttctaaaaaggttgtattagcg               tatcacgaggagggagtataa T              
The first bold T is the actA transcription start site, the last bold T is the first nt of the polio replicon
 
     The SOE PCR product was cloned upstream of the poliovirus replicon in pBHE893, replacing the actA core promoter, to construct the plasmid pIN691. The plasmid was then integrated into the genomes of select  L. monocytogenes  strains expressing holin and lysin to assess how the full-length actA promoter, with the 150 nt untranslated region, impacts the efficiency gene expression from replicons delivered to infected cells. In this embodiment, the PV replicon RNA delivered to infected cells included the 150 nt 5′ untranslated region of the ActA protein that does not constitute the authentic 5′ end of the polio virus. See Table 10 for a description of  Listeria  strains used in this study. BHK cells cultured in a 96-well plate were infected with the indicated Lm strain at an MOI=300 and stained for β-galactosidase activity at 24 hrs. post-infection. All replicons contained a β-galactosidase reporter ( FIG. 10A ). Infected cells were photographed and cells stained positive for β-galactosidase were counted using an ImmunoSpot reader ( FIG. 10B ). As shown in  FIG. 10A , when compared to the replicon in which the authentic 5′ end of the polio virus was conserved, the presence of the untranslated region of actA resulted in a greater than 10-fold decrease in the number of infected BHK cells positive for β-galactosidase expression. This data demonstrated that, in some embodiments, maintaining the authentic 5′ terminus of the PV replicon provides optimal expression of a designated antigen(s), including those related to malignant and/or infectious disease. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Lm strains for delivering a cap-independent viral-based 
               
               
                 replicon to the cytoplasm of a mammalian host cell. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Replicon 
               
               
                 Strain 
                 Background 
                 Holin/lysis 
                 Replicon 
                 promoter 
               
               
                   
               
               
                 CRS-100 
                 ΔactAΔinlB 
                 na 
                 na 
                 na 
               
               
                 Lm290 
                 ΔactAΔinlB 
                 + 
                 Sindbis virus 
                 RSV 
               
               
                 BH1147 
                 ΔactAΔinlB 
                 + 
                 Polio 
                 actA core 
               
               
                 Lm708 
                 ΔactAΔinlB 
                 + 
                 Polio 
                 actA full-length 
               
               
                   
               
            
           
         
       
     
       L. monocytogenes  induces a type I interferon (IFN) response in infected host cells upon phagosomal escape into the host cell cytoplasm. The IFN response arrests host cell protein synthesis, an effect that could diminish replicon-mediated gene expression and self-amplification when launched from  L. monocytogene.  The host cell interferon response can be inhibited by expression and secretion of one or more viral proteins known to suppress the IFN pathway. These proteins have been shown to reduce the IFN response by diverse mechanisms (see Table 11). As non-limiting examples, a selected protein(s) including those listed in the Table will be expressed from replicon-encoding  L. monocytogenes.  In some embodiments, such proteins will be expressed and secreted by  L. monocytogenes  either constituitively or induced upon infection of the host cell. In another embodiment, non-secreted variants of these proteins could be released into the infected host cell cytoplasm by co-expression of holin or holin and lysin. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Exemplary viral proteins that may be expressed by  Listeria  to 
               
               
                 mitigate effects of type I interferon. 
               
            
           
           
               
               
               
               
            
               
                 Protein 
                 Source 
                 Mechanism of Action 
                 Reference 
               
               
                   
               
               
                 TRBP 
                 human 
                 inhibition of interferon induced protein 
                 Proc Natl Acad Sci USA 1994 May 
               
               
                   
                   
                 kinase PKR 
                 24; 91(11): 4713-7 
               
               
                 Matrix 
                 VSV 
                 Blocks the nuclear pore and prevents 
                 Cancer Cell. 2003. 4: 263-75 
               
               
                 (M) 
                   
                 transport of IFN-β to the cytoplasm 
               
               
                 protein 
               
               
                 L(pro) 
                 FMDV 
                 blocks expression of type-1 IFN protein, 
                 J. Virol. 2006 Feb; 80(4): 1906-14 
               
               
                   
                   
                 reduces induction of immediate-early 
               
               
                   
                   
                 induction of IFN-beta mRNA 
               
               
                 V 
                 paramyxo 
                 interacts with mda-5 to block activity 
                 Proc Natl Acad Sci USA 2004 Dec 
               
               
                 protein 
                 viruses 
                   
                 7; 101(49): 17264-9 
               
               
                 E3L 
                 vaccinia 
                 inhibits ds-RNA-dependent protein 
                 Virology 1998 Apr 10; 243(2): 406-14 
               
               
                   
                   
                 kinase 
               
               
                 B8R 
                 vaccinia 
                 mimics IFN-gamma receptor 
                 J Gen Virol. 2002 Aug; 83(Pt8): 1953-64 
               
               
                 NS3 
                 HCV 
                 protoeolytically cleaves MAVS/IPS-1 
                 Proc Natl Acad Sci USA 2006 May 
               
               
                   
                   
                   
                 30; 103(22): 8499-504 
               
               
                 NS1 &amp; 
                 BRSV 
                 inhibits IRF-3 activation 
                 J. Virol. 2003 Aug; 77(16): 8661-8 
               
               
                 NS2 
               
               
                 N pro   
                 BVDV 
                 blocks type-1 IFN induction 
                 J. Virol. 2006 Jan; 80(2): 900-11 
               
               
                 gamma 
                 HSV-1 
                 insensitivity to alpha-IFN 
                 J. Virol. 2004 Sep; 78(18): 10193-6 
               
               
                 (1)34.5/ 
               
               
                 US11 
               
               
                 sigmaA 
                 avian 
                 blocks intracellular enzyme pathways 
                 J. Virol. 2000 Feb; 74(3): 1124-31 
               
               
                   
                 reovirus 
                 dependent on ds-RNA 
               
               
                 NS2 
                 HRSV 
                 suppresses type-1 IFN 
                 J. Virol. 2006 Jun; 80(12): 5958-67 
               
               
                 NS1 
                 influenza 
                 inhibits innate immunity by preventing 
                 J. Virol. 2006 Jul; 80(13): 6295-304 
               
               
                   
                 virus 
                 type-1 IFN release, inhibits adaptive 
               
               
                   
                   
                 immunity by attenuating DC maturation 
               
               
                 UL- 
                 HSV-2 
                 interferes with IFNa/b-mediated anti- 
                 J. Virol. 2003 Sep; 77(17): 9337-45 
               
               
                 41(vhs) 
                   
                 viral response 
               
               
                   
               
            
           
         
       
     
     An alternative means to address the IFN response entails launching a viral replicon derived from parent viruses with decreased sensitivity to the host cell interferon response. Well known to those who are skilled in the art, parent viruses with INF resistant phenotypes can be selected following serial passages in host cells capable of mounting an INF response and thus inhibiting virus replication and productive growth. Alternatively, INF resistance could be engineered in parent viruses using a reverse genetics approach in which precise mutations are introduced in the viral genome using standard molecular biology techniques. Also, portions of viral genomes from unrelated viruses to which INF-resistance has been mapped can be combined with cap-independent viral replicons to create novel cap-independent, INF-resistant chimeric replicons. 
     Example Seven 
     Utility of Recombinant  L. monocytogenes  Strains Expressing Holin, Lysin, or Holin and Lysin, for Eliciting a Specific Immune Response to an Encoded Induced Heterologous Antigen in a Vaccinated Mammal 
       FIGS. 11A and 11B  disclose Lm-induced immune response in mice. The Lm constructs were Lm-actA-OVA (no holin; no lysin) and Lm-holin-lysin-OVA (+holin; +lysin). The term “OVA” means that the Lm contained a nucleic acid encoding ovalbumin. The nucleic acid encoding ovalbumin was operably linked and in frame with actA-N 100. Transfer of ovalbumin from the  Listeria  bacterium to the environment of the host cell&#39;s cytosol was mediated by lysis of the bacterium, in the case of Lm-holin-lysin-OVA, and was mediated by a listerial secretory pathway, in the case of Lm-ActA-N100-OVA. 
       FIG. 11A  discloses that administration of both preparations of Lm resulted in ovalbumin specific immune response, where equivalent immune reponses occurred at about 1×10 6  cfu of Lm-actA-N100-OVA, and 1×10 8  cfu of Lm-holin-lysin-OVA. SIINFEKL (SEQ ID NO:53), a peptide from ovalbumin, was added to splenocytes harvested from vaccinated mice, where this addition allowing detection of any ovalbumin-specific immune response that had been produced in response to the vaccination. 
     Concomittant control immune response studies monitored immune response to a secreted protein endogenous to Lm, namely, listeriolysin O (LLO) ( FIG. 11B ). Listeriolysin expression and secretion was not expected to be influenced by the engineered nucleic acid encoding actA-N100-OVA, or by the engineered nucleic acid encoding holin-lysin-OVA. The results demonstrate that immune response to listeriolysin was equivalent where mice were administered with 1×10 6  Lm-actA-N100-OVA and 1×10 8  Lm-holin-lysin-OVA. Lower immune response against listeriolysin was found with administration of the lesser doses of Lm-holin-lysin-OVA. 
     Example Eight 
     Recombinant  Listeria monocytogenes  Strains Expressing Holin, Lysin, or Holin and Lysin, where the Heterologous Antigen does not Contain a Bacterial Secretory Peptide 
     Non-secretory Lm embodiments are provided, where the Lm contains a nucleic acid encoding a heterologous antigen, and where release of the expressed antigen from the Lm is mediated by an expressed holin. In some cases, the antigen may be a macromolecule. 
     Holin-mediated permeabilization of the listerial membrane can allow transit of an expressed heterologous antigen out of the bacterium and into an external environment, e.g., an external environment that is the cytoplasm of a mammalian host cell. 
     What is included are embodiments where greater than 10%, greater than 25%, greater than 50%, greater than 75%, and greater than 99%, of the expressed antigen is released. Also, what is included are embodiments where release is greater than 10% dependent on the expressed holin, greater than 25%, greater than 50%, greater than 75%, or greater than 99% dependent on the expressed holin. 
     In some embodiments, what is provided is Lm containing a nucleic acid encoding a heterologous antigen, and where the nucleic acid does not encode any secretory sequence. Also provided is Lm containing a nucleic acid encoding a heterologous antigen, where the nucleic acid encodes a peptide derived from a secretory sequence, but the secretory sequence is mutated to prevent secretion, or to prevent essentially all secretion. Moreover, in some embodiments, what is encompassed is a Lm containing a polynucleotide comprising a first nucleic acid encoding a heterologous antigen, where the nucleic acid does not encode any secretory sequence, and a second nucleic acid encoding a non-secretory sequence, such as groEL. 
       FIGS. 12A and 12B  disclose holin-dependent release of a fusion protein from Lm. The fusion protein did not contain any secretory sequence. The bacterium, Lm-holin, had been engineered to express holin. A plasmid encoding the fusion protein, which contained the human Mesothelin polypeptide sequence ( FIG. 12A ), was transfected into Lm-holin, and the plasmid-containing Lm-holin was used to infect mammalian cells (J774 cells). Once Lm-holin was in the J774 cells, the intracellular environment activated the actA promoter of the plasmid, resulting in production of the fusion protein. The J774 cells were incubated for 7 hours, the cells were disrupted, and soluble protein was analyzed by SDS-PAGE ( FIG. 12B ). 
     In short, the activities occurring during incubation of the J774 cells included expression of holin, expression of the fusion protein, and holin-mediated release of the fusion protein from the bacterium to the host cell&#39;s cytosol. 
     As mentioned above, after the incubation, the J774 cells were disrupted, followed by centrifugation to remove insoluble matter (including all bacteria) and separation of soluble proteins by SDS-PAGE. Mesothelin was deterred by the Western blotting method using a polyclonal antibody against Mesothelin. 
     The experiment was also conducted with control strains of Lm, that is, parental Lm (“CRS-100”) (no plasmid); Lm-holin (no plasmid); and Lm-holin-lysin (plus plasmid). 
     The following strains of  Listeria monocytogenes  were used to infect J774 cells. The lanes that are identified below and indicate the lane of the SDS-PAGE gel used to separate the expressed/release fusion protein:
     LANE ONE. Lm ΔactAΔinlB (“CRS-100”);   LANE TWO. BH543 (dnaK-hMesothelin in CRS-100);   LANE THREE. BH757 (pBHE588 in CRS-100 plus holin);   LANE FOUR. Bh759 (pBHE588 in CRS-100 plus holin and lysin).   

     The position of migration of the expressed fusion protein is shown by the arrow. The results of the gel demonstrate no expressed/released fusion protein where the bacteria were Lm ΔactAΔinlB (no plasmid), Lm ΔactAΔinlB (plus plasmid), and very slight expression/release where the bacteria were Lm ΔactAΔinlB-holin-lysin (plus plasmid). In striking contrast, dramatic expression/release occurred with Lm ΔactAΔinlB-holin (plus plasmid). 
     The results in the gel also show two non-specific bands of staining, residing above and below the Mesothelin band. 
     The results demonstrate that Lm-holin is an effective agent for expressing and releasing polypeptides into a host cell, even where the polypeptide is not a secretory protein. Without implying any limitation on the present invention, the results also demonstrate that in this instance, Lm-holin-lysin was a relatively poor agent for expressing and releasing the indicated polypeptide into the host cell. 
     What is provided is Lm-holin, vaccines comprising Lm-holin, and related methods. These methods include using Lm-holin for the intracellular expression and release of any substance, e.g., a water-soluble substance, a macromolecule, a polypeptide, a heterologous antigen, a tumor antigen, an infectious agent antigen, a complex including a polypeptide, a nucleic acid, a plasmid, a viral-based expression cassette, a ssRNA (positive strand) viral-based expression cassette, and the like. 
     In some embodiments, what is also provided is Lm-holin that does not contain any recombinant nucleic acid encoding a lysin, vaccines comprising Lm-holin that does not contain a recombinant nucleic acid encoding a lysin, and related methods. These methods include using the Lm-holin that does not contain any recombinant nucleic acid encoding a lysin, for the expression and release of any substance, e.g., a water-soluble substance, a macromolecule, a polypeptide, a heterologous antigen, a tumor antigen, an infectious agent antigen, a complex including a polypeptide, a nucleic acid, a plasmid, a viral-based expression cassette, a ssRNA (positive strand) viral-based expression cassette, and the like. 
     Moreover, in some embodiments, what is provided is a method for stimulating immune response against a heterologous antigen, comprising administering a Lm-holin-heterologous antigen where the stimulated immune response is greater than that obtainable with administering a suitable control Lm that lacks a nucleic acid encoding holin, where the greater is at least 20% greater; 50% greater; 100% greater (2-fold greater); 5-fold greater; 10-fold greater, or more. A suitable control is Lm-heterologous antigen (no holin). 
     Also, what is encompassed is a method for stimulating immune response against a heterologous antigen, comprising administering a Lm-holin-heterologous antigen (lacking a recombinant nucleic acid encoding a lysin) where the stimulated immune response is greater than that obtainable with administering a suitable control Lm that lacks a nucleic acid encoding holin, where the greater is at least 20% greater; 50% greater; 100% greater (2-fold greater); 5-fold greater; 10-fold greater, or more. A suitable control is Lm-heterologous antigen that lacks a recombinant nucleic acid encoding a lysin (no holin). 
     The Lm of the invention can contain a more than one nucleic acids, each encoding the same holin, each encoding a different holin, or any combination thereof. The nucleic acids can be operably linked with a promoter specifically activated by an environment found inside a host mammalian cell, e.g., prfA promoter, any prfA-activatable promoter, actA promoter, promoters sensitive to low iron concentrations, and so on. Constitutively active promoters are also available. The promoter can also be one specifically used by a particular RNA polymerase, where this RNA polymerase is expressed (or activated) in increased amounts by the Lm when the Lm is inside a mammalian host cell. 
     Experimental details were as follows. These details are not intended to limit the invention. In order to test the utility of holin-lysin strains for delivering antigens lacking a bacterial secretory peptide, the following plasmids were engineered. A human mesothelin ORF optmized for expression in  Listeria  was synthesized (CCN16543, Blue Heron Biotechnology) and used as template for PCR with the following primer set: 
     
       
         
           
               
            
               
                 PL339 (forward): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 54) 
               
            
           
           
               
            
               
                 5′ GGGCGGCCGCGAGCTCTTAGCCTTGTAAACCTAAACCTAATGTATC 
               
               
                 TAA 3′ 
               
               
                   
               
               
                 PL340 (reverse): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 55) 
               
            
           
           
               
            
               
                 5′ GGGGATCCCGTACATTAGCAGGTGAAACAGGTCAAGAA 3′ 
               
            
           
         
       
     
     The PCR product (huMesothelin Δsignal sequence Δgpi anchor) was purified over a Qiagen® column and digested with BamHI and EagI. The pINT vector containing the actA promoter, pBHE135, was digested with the same set of restriction enzymes, treated with CIP and purified over a Qiagen® column. The vector and insert were ligated together using T4 DNA ligase. Chloramphenicol resistant colonies were screened by PCR and confirmed by restriction digest, resulting in pBHE139. 
     The groEL ORF was PCR amplified from DP-L4056 genomic DNA using the following primer set: 
     
       
         
           
               
               
            
               
                   
                 PL714 (forward): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 56) 
               
            
           
           
               
               
            
               
                   
                 5′ AAAATCGATATGAGCAAAATTATCGGAATTGACTTA 3′ 
               
               
                   
                   
               
               
                   
                 PL715 (reverse): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 57) 
               
            
           
           
               
               
            
               
                   
                 5′ AAAGGATCCTTTGTTTTCTTTGTCGTCGTCATTTAC 3′ 
               
            
           
         
       
     
     The PCR product was purified over a Qiagen® column and digested with ClaI and BamHI. The plasmid pBHE139 was digested with the same set of restriction enzymes, treated with CIP (NEB) and purified over a Qiagen® column. Vector and insert were ligated together using T4 DNA ligase. Chloramphenicol resistant colonies were screened by PCR and positive clones confirmed by restriction digest. This resulted in the plasmid pBHE558. This plasmid was transferred to  Listeria  strain CRS 100 by conjugation (Lauer et al., supra) resulting in the erythromycin resistant strain BH543. This strain was cured of vector backbone sequences (ENGINEERED  LISTERIA  AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197), resulting in the erythromycin sensitive strain BH755. 
     To engineer holin and holin-lysin expressing variants of the groEL-huMesothelin strain, BH755 was conjugated with SM10 cells containing either pBHE633 (actAp_holin directed to comK locus) or pBHE636 (actAp_holin-lysin directed to comK locus). The selection of erythromycin resistant colonies resulted in BH757 (holin only) and BH759 (holin-lysin) expressing variants. Levels of expression and secretion of the groEL-huMesothelin protein was analyzed by Western blot in both broth and in mammalian host cells. 
     Example Nine 
     Recombinant  Listeria monocytogenes  Strains Expressing Holin, or Holin and Lysin, where the Nucleic Acid Encoding Holin is not from a Listeriophage or from a Listerial Genus 
     What is provided is a  Listeria monocytogenes  bacterium containing a nucleic acid encoding a holin that is not listerial and not from a listeriophage. The nucleic acid can be from a bacteriophage that is not a listeriophage, from a bacterium that is not listerial, or from other organisms. 
     The invention encompasses non-listeriophage holins, including  Serratia marcescens  NucE (Berkmen, et al. (1997) 179:6522-6524);  Staphylococcus aureus  bacteriophage 187 holin (Loessner, et al. (1999) J. Bacteriol. 181:4452-4460; phage lambda holins and  Lactobacillus gasseri  phi-adh holin (Henrich, et al. (1995) J. Bacteriol. 177:723-732; phage phi-29 holin (Steiner, et al. (1993) J. Bacteriol. 175:1038-1042);  Bacillus  phage PZA holin (Loessner, et al. (1997) J. Bacteriol. 179:2845-2851); phage T4 gpt holin (Dressman and Drake (1999) J. Bacteriol. 181:4391-4396); phage PRD1 holin (Ziedaite, et al. (2005) J. Bacteriol. 187:5397-5405);  Borrelia burgdorferi  prophage BIyA (Damman, et al. (2000) J. Bacteriol. 182:6791-6797);  Bacillus subtilis  ywcE holin (Real, et al. (2005) J. Bacteriol. 187:6443-6453);  Staphylococcus aureus  lrgA holin and cidA holin (Brunskill and Bayles (1996) J. Bacteriol. 178:5810-5812; Rice, et al. (2004) J. Bacteriol. 186:3029-3037);  Streptococcus pneumoniae  cph1 holin, pneumococcal phage EJ-1 holin, phi-LC3 holin and Tuc2009 holin of  Lactococcus lactis  phage (Martin, et al. (1998) J. Bacteriol. 180:210-217); bacteriophage P2 gene Y holin (Ziermann, et al. (1994) J. Bacteriol. 176:4974-4984); and bacteriophage PRD1 holin P35 (Rydman and Bamford (2003) J. Bacteriol. 185:3795-3803). 
     Nucleic acids in bacterial genomes, encoding proteins identified as holins and holin-like proteins, are available (Table 12). Some of these holins can be characterized as bacterial holins, and not as holins of cryptic phages, i.e., phage genomes integrated in the bacterial genome. These holin genes include the CidA gene of  S. aureus  (see, e.g., Rice, et al. (2003) J. Bacteriol. 185:2635-2643; Rice and Bayles (2003) Mol. Microbiol. 50:729-738; Bayles (2000) Trends Microbiol. 8:274-278; GenBank Acc. No. AY581892). 
     In some embodiments, what is also contemplated are listerial strains that are not Lm, for example,  L. innocua  engineered to contain factors that mediate entry into antigen presenting cells, and that mediate exit from the phagolysosome to the host cell&#39;s cytoplasm. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Bacterial genomic nucleic acids encoding holins. 
               
            
           
           
               
               
            
               
                 Bacterium 
                 GenBank Acc. No. 
               
               
                   
               
               
                 
                   Bacillus subtilis 
                 
                 Z99117, nt 51006-51428. 
               
               
                 
                   Bacillus subtilis 
                 
                 NC_000964, nt 2263876-2264088; 3932232-3932618. 
               
               
                   Bacillus anthracis  strain Sterne 
                 NC_005945, five holins, e.g., 3432919-3433284. 
               
               
                 
                   Pseudomonas entomophila 
                 
                 NC_008027, compl. nt 4463886-4464239. 
               
               
                 
                   Escherichia coli 
                 
                 BA000007, ten holins, e.g., nt 901806-902021. 
               
               
                   Listeria monocytogenes  strain 
                 NC_002973 nt 142006-142428. 
               
               
                 4b F2365 
               
               
                 
                   Listeria innocua 
                 
                 AL596169, nt compl. 165378-165638. 
               
               
                 
                   Staphylococcus epidermidis 
                 
                 CP000029, nt 2047024-2047482. 
               
               
                 
                   Erwinia carotovora 
                 
                 NC_004547, compl. nt 2950159-2950467. 
               
               
                 
                   Corynebacterium diphtheriae 
                 
                 NC_002935, six holins, e.g., nt 3637616-3637981. 
               
               
                 
                   Corynebacterium diphtheriae 
                 
                 BX248360, compl. nt 129273-129650. 
               
               
                 
                   Staphylococcus aureus 
                 
                 AJ938182, four holins, e.g., compl. 1846356-1846793. 
               
               
                 
                   Salmonella typhimurium 
                 
                 AE008823 nt 16310-16529. 
               
               
                 
                   Rhodopseudomonas palustris 
                 
                 BX572594, nt 258068-258451. 
               
               
                   
               
            
           
         
       
     
     Example Ten 
     Tables Identifying Exemplary strains of  Listeria monocytogenes  and Plasmid Constructs 
       
     
       
         
           
               
             
               
                 TABLE 13A 
               
               
                   
               
               
                 Plasmid Constructions 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 pSH263 
                 pSH252 with Sindbis virus/lacZ replicon 
               
               
                   
                 downstream of RSV promoter 
               
               
                 pSH252 
                 pAM401 with oriT for transfer from  E. coli  donor to 
               
               
                   
                   Listeria  by conjugation. 
               
               
                 Sinrep/lacZ 
                 Sindbis virus replicon with lacZ reporter 
               
               
                 Sinrep21 
                 Sinbis virus replicon with RSV promoter for DNA 
               
               
                   
                 launch 
               
               
                 pINT (aka p217) 
                 Integration vector and source of oriT 
               
               
                 pAM401 
                   E. coli / L. monocytogenes  shuttle plasmid 
               
               
                 pSH258 
                 pSH252 with BamHI-EagI fragment from Sinrep21 
               
               
                 pBHE530 
                 pAM-CMV-lacZ 
               
               
                 pJ10:4934 
                 Sindbis virus DI/IRES 5′end 
               
               
                 pCO330 
                 Sinrep DI/IRES intermediate 
               
               
                 pCO390 
                 Sinrep DI/IRES replicon 
               
               
                 pIN548 
                 pINT containing actA core promoter 
               
               
                 pIN586 
                 PIN548 with 5′ end of polio replicon including 
               
               
                   
                 IRES, VP4, 2A cleavage site 
               
               
                 pIN599 
                 pIN586 with 3′ end of polio replicon including 
               
               
                   
                 nonstructural viral proteins and poly A tail 
               
               
                 pIN662 
                 pIN599 with complete polio replicon functionally 
               
               
                   
                 linked to actA core promoter. 
               
               
                 pBHE893 
                 pIN662 with SL8-b galactosidase fusion 
               
            
           
           
               
            
               
                 Bacteria Strains 
               
            
           
           
               
               
            
               
                 SM10 
                   E. coli  conjugation donor strain 
               
               
                 B-Ec-266 
                 SM10 containing pSH263 
               
               
                 4029uvr 
               
               
                 DP-L4056 
               
               
                 BH334 
                 4056 holin 
               
               
                 BH336 
                 4056 lysin 
               
               
                 BH276 
                 4056 holin-lysin 
               
               
                 B-Lm-274 
                 4056 containing pSH263 
               
               
                 B-Lm-276 
                 BH336 with pSH263 
               
               
                 B-Lm-277 
                 BH334 with pSH263 
               
               
                 B-Lm-278 
                 BH276 with pSH263 
               
               
                 BH276(pBHE530) 
                 BH276 with pBHE530 
               
               
                 BH727 
                 4056 with two copies of holin 
               
               
                 B-Lm-284 
                 4029uvr::pBHE292, 4029uvr holin-lysin 
               
               
                 B-Lm-288 
                 B-Lm-284 with pSH263 
               
               
                 B-Lm-289 
                 4029uvr with pSH263 
               
               
                 B-Lm-414 
                 BH727 with pSH263 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13B 
               
             
            
               
                   
               
               
                 Strains of  Listeria monocytogenes . 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Site of integration 
                   
               
               
                 Strain 
                 Derived from 
                 Plasmid 
                 or episomal 
                 Features 
               
               
                   
               
               
                 ActAN100_OVA(AH1A5) 
                 CRS-100 
                 none 
                 n/a 
                 Contains N- 
               
               
                   
                   
                   
                   
                 terminal fusion 
               
               
                   
                   
                   
                   
                 of first 100 aa of 
               
               
                   
                   
                   
                   
                 ActA to 
               
               
                   
                   
                   
                   
                 OVA(AH1A5) 
               
               
                   
                   
                   
                   
                 under the control 
               
               
                   
                   
                   
                   
                 of the actA 
               
               
                   
                   
                   
                   
                 promoter 
               
               
                   
                   
                   
                   
                 at ActA locus 
               
               
                 CRS100 
                 DP-L4056 
                 none 
                 n/a 
                 deleted ActA, 
               
               
                   
                   
                   
                   
                 deleted InlB 
               
               
                 DP-L4027 
                 DP-L4056 
                 none 
                 n/a 
                 deleted hly, 
               
               
                   
                   
                   
                   
                 cannot escape 
               
               
                   
                   
                   
                   
                 from 
               
               
                   
                   
                   
                   
                 phagolysosome 
               
               
                 DP-L4056 
                 10403S 
                 none 
                 n/a 
                 Phage cured 
               
               
                 BH225 
                 CRS-100 
                 pBHE292 
                 tRNA arg   
                 PSA holin-lysin 
               
               
                   
                   
                   
                   
                 under the control 
               
               
                   
                   
                   
                   
                 of actA 
               
               
                   
                   
                   
                   
                 promoter 
               
               
                 BH226 
                 CRS-100 
                 pBHE292 
                 tRNA arg   
                 PSA holin-lysin 
               
               
                   
                   
                   
                   
                 under the control 
               
               
                   
                   
                   
                   
                 of actA 
               
               
                   
                   
                   
                   
                 promoter 
               
               
                 BH276 
                 DP-L4056 
                 pBHE292 
                 tRNA arg   
                 PSA holin-lysin 
               
               
                   
                   
                   
                   
                 under the control 
               
               
                   
                   
                   
                   
                 of actA 
               
               
                   
                   
                   
                   
                 promoter 
               
               
                 BH279 
                 ActAN100_OVA 
                 pBHE292 
                 tRNA arg   
                 PSA holin under 
               
               
                   
                 (AH1A5) 
                   
                   
                 the control of 
               
               
                   
                   
                   
                   
                 actA promoter 
               
               
                 BH334 
                 DP-L4056 
                 pBHE340 
                 tRNA arg   
                 PSA holin under 
               
               
                   
                   
                   
                   
                 the control of 
               
               
                   
                   
                   
                   
                 actA promoter 
               
               
                 BH336 
                 DP-L4056 
                 pBHE361 
                 tRNA arg   
                 PSA lysin under 
               
               
                   
                   
                   
                   
                 the control of 
               
               
                   
                   
                   
                   
                 actA promoter 
               
               
                 BH561 
                 BH225 
                 pBHE292(cured) 
                 tRNA arg   
                 Erm S  derivative 
               
               
                   
                   
                   
                   
                 of BH225 
               
               
                 BH567 
                 BH334 
                 pBHE340(cured) 
                 tRNA arg   
                 Erm S  derivative 
               
               
                   
                   
                   
                   
                 of BH334 
               
               
                 BH721 
                 DP-L4056 
                 pBHE631 
                 comK 
                 contains 
               
               
                   
                   
                   
                   
                 luciferase under 
               
               
                   
                   
                   
                   
                 the control of the 
               
               
                   
                   
                   
                   
                 actA promoter 
               
               
                   
                   
                   
                   
                 with an 
               
               
                   
                   
                   
                   
                 intervening 
               
               
                   
                   
                   
                   
                 IRES 
               
               
                 BH727 
                 BH567 
                 pBHE633 
                 comK 
                 contains two 
               
               
                   
                   
                   
                   
                 copies of holin 
               
               
                   
                   
                   
                   
                 under the control 
               
               
                   
                   
                   
                   
                 of actA 
               
               
                   
                   
                   
                   
                 promoter, one at 
               
               
                   
                   
                   
                   
                 tRNA arg  and the 
               
               
                   
                   
                   
                   
                 other at comK 
               
               
                 BH741 
                 BH721 
                 pBHE631(cured) 
                 comK 
                 Erm S  derivative 
               
               
                   
                   
                   
                   
                 of BH721 
               
               
                 BH743 
                 BH741 
                 pBHE633 
                 comK 
                 contains 
               
               
                   
                   
                   
                   
                 luciferase under 
               
               
                   
                   
                   
                   
                 the control of the 
               
               
                   
                   
                   
                   
                 actA promoter 
               
               
                   
                   
                   
                   
                 with an 
               
               
                   
                   
                   
                   
                 intervening 
               
               
                   
                   
                   
                   
                 IRES at tRNA arg , 
               
               
                   
                   
                   
                   
                 holin under 
               
               
                   
                   
                   
                   
                 control of actA 
               
               
                   
                   
                   
                   
                 promoter at 
               
               
                   
                   
                   
                   
                 comK 
               
               
                 BH745 
                 BH741 
                 pBHE636 
                 comK 
                 contains 
               
               
                   
                   
                   
                   
                 luciferase under 
               
               
                   
                   
                   
                   
                 the control of the 
               
               
                   
                   
                   
                   
                 actA promoter 
               
               
                   
                   
                   
                   
                 with an 
               
               
                   
                   
                   
                   
                 intervening 
               
               
                   
                   
                   
                   
                 IRES at tRNA arg , 
               
               
                   
                   
                   
                   
                 holin-lysin 
               
               
                   
                   
                   
                   
                 under control of 
               
               
                   
                   
                   
                   
                 actA promoter at 
               
               
                   
                   
                   
                   
                 comK 
               
               
                 BH575 
                 DP-L4056 
                 pBHE573 
                 episomal 
                 allows delivery 
               
               
                   
                   
                   
                   
                 of luciferase 
               
               
                   
                   
                   
                   
                 based eukaryotic 
               
               
                   
                   
                   
                   
                 expression 
               
               
                   
                   
                   
                   
                 cassette from 
               
               
                   
                   
                   
                   
                 WT  Listeria  to 
               
               
                   
                   
                   
                   
                 infected host 
               
               
                   
                   
                   
                   
                 cells 
               
               
                 BH577 
                 BH276 
                 pBHE573 
                 episomal 
                 allows delivery 
               
               
                   
                   
                   
                   
                 of luciferase 
               
               
                   
                   
                   
                   
                 based eukaryotic 
               
               
                   
                   
                   
                   
                 expression 
               
               
                   
                   
                   
                   
                 cassette from 
               
               
                   
                   
                   
                   
                 holin-lysin 
               
               
                   
                   
                   
                   
                 expressing 
               
               
                   
                   
                   
                   
                   Listeria  to 
               
               
                   
                   
                   
                   
                 infected host 
               
               
                   
                   
                   
                   
                 cells 
               
               
                 BH579 
                 BH334 
                 pBHE573 
                 episomal 
                 allows delivery 
               
               
                   
                   
                   
                   
                 of luciferase 
               
               
                   
                   
                   
                   
                 based eukaryotic 
               
               
                   
                   
                   
                   
                 expression 
               
               
                   
                   
                   
                   
                 cassette from 
               
               
                   
                   
                   
                   
                 holin expressing 
               
               
                   
                   
                   
                   
                   Listeria  to 
               
               
                   
                   
                   
                   
                 infected host 
               
               
                   
                   
                   
                   
                 cells 
               
               
                 BH581 
                 BH336 
                 pBHE573 
                 episomal 
                 allows delivery 
               
               
                   
                   
                   
                   
                 of luciferase 
               
               
                   
                   
                   
                   
                 based eukaryotic 
               
               
                   
                   
                   
                   
                 expression 
               
               
                   
                   
                   
                   
                 cassette from 
               
               
                   
                   
                   
                   
                 lysin expressing 
               
               
                   
                   
                   
                   
                   Listeria  to 
               
               
                   
                   
                   
                   
                 infected host 
               
               
                   
                   
                   
                   
                 cells 
               
               
                   
               
            
           
         
       
     
     All publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.