WO2005089101A2 - Non-pathogenic listeria vaccine vectors and methods of treatment therewith - Google Patents

Abstract

The present invention relates to vaccine vectors comprising recombinant strains of Listeria, e.g., L. innocua. In one aspect, the invention relates to vaccine vectors comprising a recombinant Listeria innocua strain, where the recombinant Listeria innocua strain comprises one or more heterologous genes, and where the recombinant Listeria innocua strain is capable of eliciting an immunogenic response in a subject. In one embodiment, the heterologous gene(s) are derived from Listeria monocytogenes, e.g., the hly gene, the inlA gene, and/or the prfA gene. In one embodiment, the recombinant Listeria vaccine vectors of the invention encode a heterologous antigen, e.g., a viral protein, a bacterial protein, a fungal protein, a parasitic protein, a glycoprotein, a lipoprotein, and/or a glycolipid. The invention provides methods for treating and/or preventing infections, diseases or disorders in a subject by administering the recombinant Listeria vaccine vectors of the invention to the subject.

Description

NON-PATHOGENIC LISTERIA VACCINE VECTORS AND METHODS OF TREATMENT THEREWITH

Realted Applications This application claims priority to U.S. Provisional Application No.

60/541708 filed on February 3, 2004, incorporated herein in its entirety by reference.

Background of the Invention The use of vaccines is an efficient and cost-effective medical tool for the management of infectious diseases, including infectious diseases caused by bacteria, viruses, parasites, and fungi. ?fn addition to effecting protection against infectious diseases, vaccines may now also be developed which stimulate the host's immune system to intervene in tumor growth. Furthermore, there is an increasing need for vaccines against infectious agents which may be used in bioterrorism attacks. Host immune responses include both the humoral immune response involving antibody production and the cell-mediated immune response. Protective immunization via vaccine has usually been designed to induce the formation of humoral antibodies directed against infectious agents, tumor cells, or the action of toxins. However, the control of certain diseases characterized by the presence of tumor cells or by chronic infection of cells with infectious agents often requires a cell-mediated immune response either in place of, or in addition to, the generation of antibodies. While the humoral immune response may be induced by using live infectious agents and agents which have been inactivated, a cellular immune response is most effectively induced through the use of live agents as vaccines. Such live agents include live infectious agents which may gain access to the cytoplasm of host cells where the proteins encoded by these agents are processed into epitopes which when presented to the cellular immune system, induce a protective response. A number of bacteria with varying degrees of virulence form the genus Listeria. These organisms are facultatively anaeorbic, non-spore-forming Gram-positive rods. Listeria monocytogenes (L. monocytogenes) is the prototypic intracellular bacterial pathogen which elicits a predominantly cellular immune response when inoculated into an animal (Kaufmami, (1993), Ami. Rev. Immunol. 11:129-163). L. monocytogenes can be found in natural sources such as soil, especially in decaying plants, as well as in sewage and river sludge. Listeria species contaminate vegetables, dairy products, meat, and poultry. L. monocytogenes can cause listeriosis, a serious food-borne disease, in humans and animals. E. monocytogenes is most frequently diagnosed during pregnancy and is associated with neonatal morbidity and mortality, even though E. monocytogenes infection rarely threatens the life of the mother (Gellin and Broome, (1989) JAMA 261(9):1313). Aside from neonates, L. monocytogenes most frequently causes serious infections in immunosuppressed individuals, especially in lymphoma patients treated with steroids and undergoing chemotherapy, and in the elderly. Whole clinical manifestations in all species can vary. ?bι humans and in nonhuman primates, three major forms of listeriosis are recognized: 1) abortion or septicemia shortly after birth; 2) meningoencephalitis; and 3) septicemia. The primary route of L. monocytogenes transmission to vertebrate hosts in through the oral route via contaminated food, and the intestine is considered to be the first site of colonization. When used as a vector for the transmission of genes encoding heterologous antigens derived from infectious agents or derived from tumor cells, recombinant Listeria monocytogenes encoding and expressing the heterologous antigen have been shown to successfully protect mice against challenge by lymphocytic choriomeningitis virus (Shen et al. (1995), Proc. Nat/. Acad. Sci. USA 92:3987-3991; Goossens et al. (1995), Int. Immunol. 7:797-802) and influenza virus (LCMN) (Ikonomidis et al. (1997) Vaccine 15:433-440). Further, heterologous expressing recombinant Listeria monocytogenes have been used to protect mice against lethal tumor cell challenge (Pan et al. (1995) Nat. Med. 1:471-477; Paterson and Ikonomidis (1996) Curr. Opin. Immunol. 8:664-669). ?In addition, it is known that a strong cell-mediated immune response directed against HIN-1 gag protein may be induced in mice vaccinated with a gαg-encoding, attenuated Listeria monocytogenes vector (Frankel et al. (1995) J. Immunol. 155:4775-4782). Although the potential broad use of Listeria monocytogenes as a vaccine vector for the prevention and treatment of infectious disease and cancer has significant advantages over other vaccines, the issue of safety during use of Listeria monocytogenes is not trivial. L. monocytogenes, which is the most common strain of Listeria, is accompanied by potentially severe side effects, including the development of listeriosis in the inoculated animal. A need thus remains for an improved vaccine vector. Summary of the Invention The present invention relates to vaccines comprising recombinant strains of Listeria, e.g., L. innocua, referred to herein as "recombinant Listeria vaccine vectors." In one aspect, the invention relates to vaccines comprising a recombinant L. innocua strain, where the recombinant Listeria innocua strain comprises one or more heterologous genes, and where the recombinant E. innocua strain is capable of eliciting an immunogenic response in a subject to the inserted heterologous gene(s). In one embodiment, the heterologous gene or genes are derived from L. monocytogenes. In another embodiment, the heterologous gene is the hly gene. In still another embodiment, the heterologous genes are the hly gene the inlA gene. In a further embodiment, the heterologous genes are the hly gene, the inlA gene, and a gene encoding a transcription factor, e.g., the prfA gene. In another aspect of the invention, administering the recombinant L. innocua strain causes the production of an immune response, e.g., a cytotoxic T-cell immune response in a subject, e.g., a human. In one embodiment, the recombinant Listeria vaccine vectors of the invention further comprise DNA encoding one or more heterologous antigens. In another embodiment, the vaccines of the invention comprise a vector comprising a DNA encoding one or more heterologous antigens. In one embodiment, the antigen may be selected from the group consisting of: a viral protein, a bacterial protein, a fungal protein, a parasite protein, a glycoprotein, a lipoprotein, or a glycolipid. In another embodiment, the antigen is selected from the group consisting of: Εbola virus, HIV, SARS, a small pox antigen, hepatitis A, B or C virus, human rhino virus, Herpes simplex virus, poliovirus (type 2 or type 3), foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), the Ε7 protein of HPV, and fragments containing the E7 protein or its epitopes, simian immunodeficiency virus (SIN), malarial antigens, fungal antigens, bacterial antigens, tumor antigens, antigens derived from Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, and E. coli antigens, Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens), and antigens of parasites including flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria. In another aspect, the invention pertains to methods of eliciting an immune response, e.g., a cytotoxic T-cell immune response, to an antigen of an infectious agent in a subject, e.g., a human subject, comprising administering to the subject an effective amount of a vaccine comprising recombinant L. innocua, where the recombinant E. innocua comprises one or more heterologous genes. In one embodiment, the vaccine is administered orally. In another embodiment, the vaccine is administered in multiple doses. In still another embodiment, the recombinant L. innocua further comprises DNA encoding a heterologous antigen. In still another embodiment, the recombinant L. innocua further comprises a vector comprising a DNA encoding a heterologous antigen. In yet another aspect, the invention relates to methods of treating or preventing an infection, disease or disorder in a subject comprising administering to the subject a vaccine comprising a recombinant E. innocua strain, wherein the recombinant Listeria strain comprises one or more heterologous genes, thereby treating or preventing an infection, disease or disorder in the subject, e.g., a human, hi one embodiment, the infection, disease, or disorder is selected from the group consisting of: any infection, disease or disorder caused by or related to a fungus, parasite, virus, or bacteria, listeriosis, Εbola virus, SARS, small pox, hepatitis A, hepatitis B, hepatitis C, diseases and disorders caused by human rhinovirus, HIV and AIDS, Herpes, polio, foot-and- mouth disease, rabies, diseases or disorders caused by or related to: rotavirus, influenza, coxsackie virus, human papilloma virus, SIV, malaria, cancer, e.g., tumors, and diseases or disorders caused by or related to infection by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, E. coli, flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites.

Brief Description of the Figures Figure 1 depicts the construction of vectors included in the present invention. Figure 2 depicts the nucleotide sequence of the hly gene (SΕQ ID NO: 1). Figure 3 depicts the nucleotide sequence of the inlA gene (SΕQ ID NO:2). Figure 4 depicts the nucleotide sequence of theprfA gene (SΕQ ID NO:3).

Detailed Description The present invention relates to vaccines comprising recombinant strains of

Listeria, e.g., L. innocua, referred to herein as "recombinant Listeria vaccine vectors." The recombinant Listeria vaccine vectors of the invention comprise E. innocua which has been modified to include one or more heterologous bacterial genes, e.g., genes wliich are contained within the E. monocytogenes genome but which are not contained within the wild-type L. innocua genome, either alone or in combination with other heterologous genes. The heterologous gene or genes introduced into the E. innocua genome function to confer immunogenicity but do not confer pathogenicity, e.g., the gene or genes allow host cell entry and development of an immune response, but do not allow cell to cell spread of the bacteria. The genes or genes which are inserted into the L. innocua genome to create the recombinant Listeria vaccine vectors of the invention may include certain heterologous genes contained within the virulence gene cluster of L. monocytogenes, alone or in combination with one or more additional heterologous genes. The term "heterologous gene," as used herein, refers to any gene which is not normally expressed in wild-type E. innocua. For example, the hly and prf. ^"A genes are heterologous genes which may be inserted into E. innocua. The genomes of L. innocua and L. monocytogenes have been sequenced and found to be strikingly similar. However, the genome of L. innocua does not contain a "hly virulence gene cluster" which confers pathogenicity to the L. monocytogenes strain (Glaser, et al. (2001) Science 264:849-852). The hly virulence gene cluster is approximately 9kb and encodes six (6) genes: prf A, plcA, hly, mpl, act A andplcB. hly encodes listeriolysin O (LLO), which is a pore-forming toxin required to disrupt the pkagocytic vacuole and release bacteria into the cytoplasm, a prerequisite for intracellular proliferation of the bacteria, act A encodes the surface protein ActA, which is responsible for actin-based motility and cell-to-cell spread. prfA encodes a transcriptional factor that activates the expression of all genes of the cluster as well as two genes, internalin A (encoded by inlA) and internalin B (encoded by MB), which reside at a different locus. PlcA, PlcB and Mpl cooperate with LLO in the disruption of the primary vacuoles after the phagocytosis of extracellular Listeria. Accordingly, the virulence gene cluster is essential for pathogenicity. The genes contained within the Listeria virulence gene cluster, and their functions, are listed in Table 1. Table 1. Genes Contained within the Listeria Virulence Gene Cluster

*For review, see Vazquez-Boland, et al. (2001) Clin. Microbiol. Rev. 14(3):584; Vazquez-Boland, et al. (2001) Microbes Infect. 3(7):571; and Glaser et al. (2001) Science 294(5543):849. Each of the references contained in Table 1 are expressly incorporated herein by reference.

In one embodiment, the recombinant Listeria vaccine vectors of the invention are produced by inserting the hly gene into the non-invasive, non-pathogenic L. innocua genome. In another embodiment, the recombinant Listeria vaccine vectors of the invention comprise the hly gene and the inlA gene. In still another embodiment, the recombinant Listeria vaccine vectors of the invention comprise the hly gene, the inlA gene, and a transcription factor, e.g., the prfA gene. Because actA and other genes included in the virulence cluster are missing from the recombinant L. innocua, these vectors are be non-pathogenic or less pathogenic than previously described attenuated L. monocytogenes wherein actA and plcB are deleted (Angelakopoulos et al. (2002) Infection and Immunity 70(7):3592-3601). Additional potential L. monocytogenes genes which may be inserted into the L. innocua genome for the production of recombinant Listeria vaccine vectors of the invention include genes which are not present in the L. innocua genome but which do not confer pathogenicity into E. innocua. The complete genome of L. monocytogenes (ΕGD-e strain) is set forth as GenBank Accession No. AL591824 (gi:30407125) and described in Glaser, et al. (2001) Science 294(5543): 849-52). The complete genome of L. innocua (CLIP 11262 strain) is set forth as GenBank Accession No. AL592022 (gi: 30407126). The L. innocua strain also contains aplasmid set forth as GenBank Accession No. AL592102 (gi:16415787). The invention should not be construed as being limited solely to the use of a Listeria species having the sequence as set forth in GenBank Accession No. AL592022, i.e., the L. innocua species. It will be appreciated that genes may be deleted from any species of Listeria to, e.g., render the strain non-pathogenic while retaining immunogenicity.

Accordingly, the invention should therefore be construed to include any and all Listeria species wliich have substantial homology to the L. innocua species shown in GenBank Accession No. AL592022. Preferably, the genome of the Listeria species about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the L. innocua species having the sequence as set forth in GenBank Accession No. AL592022. The present invention is based, at least in part, on the discovery that the administration of a recombinant Listeria vaccine vector is useful for the prevention and/or treatment of infection caused by Listeria. Furthermore, it has been discovered that the administration of a recombinant Listeria vaccine vector encoding and expressing a heterologous antigen is useful for the prevention and/or treatment of infection caused by the organism from which the heterologous antigen is derived. The heterologous antigen may be from of any number of infectious agents, including, but not limited to, ΕBOV, HIV, influenza, smallpox, and mycobacteria. The administration of a recombinant Listeria vaccine vector of the present invention may be also useful for the prevention and/or treatment of tumor growth or metastasis in a subject, e.g., a mammal. The term "vaccine," as used herein, refers to a recombinant Listeria strain of the invention which when inoculated into a mammal has the effect of stimulating an immune response such as a cellular immune response comprising a T cell response. The T cell response may be a cytotoxic T cell response directed against macromolecules produced by the bacteria. However, the induction of a T cell response comprising other types of T cells by the vaccine of the invention is also contemplated. For example, Listeria infection also induces both CD4+ T cells and CD8+ T cells. Induced CD4+ T cells are responsible for the synthesis of cytokines, such as interferon-γ, IL-2 and TNF- . CD8+ T cells may be cytotoxic T cells and also secrete cytokines such as interferon-γ and T?NF-O All of these cells and the molecules synthesized therein play a role in the infection and subsequent protection of the host against Listeria. Cytokines produced by these cells activate additional T cells and also macrophages and recruit polymorphonuclear leukocytes to the site of infection. The invention includes vectors which comprise an isolated nucleic acid encoding heterologous genes including, for example, hly, inlA, and/or prfA. The term "isolated nucleic acid molecule" includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The isolated nucleic acids of the invention should be construed to include an RNA or a DNA sequence specifying the hly, inlA, and/or prfA genes, and any modified forms thereof, including chemical modifications of the DNA or RNA which render the nucleotide sequence more stable when it is cell free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleotide sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleotide sequences are contemplated in the present invention. The invention should not be construed as being limited solely to the DNA and amino acid sequences shown in Figure 2. Once armed with the present invention, it is readily apparent to one skilled in the art that any other DNA and encoded amino acid sequence of the hly, inlA, and/or prfA genes of other Listeria species maybe obtained by following the procedures described herein and known in the art. The invention should therefore be construed to include any and all hly, inlA, and/or prfA DNA sequence and conesponding amino acid sequence, having substantial homology to the hly, inlA, and/or prfA DNA sequence, and the conesponding amino acid sequence, shown in Figure 2. Preferably, DNA which is substantially identical is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the hly, inlA, and r prfA sequence shown in Figure 2. Preferably, an amino acid sequence which is substantially identical is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences encoded by the hly, inlA, and/or prfA genes shown in Figure 2. The recombinant Listeria vaccine vectors of the invention may encode one or more heterologous antigens and therefore may treat or prevent one or more diseases or disorders, i.e., multiple-antigen vaccines. In addition, more than one recombinant Listeria vaccine vector encoding different antigens may be administered to a subject simultaneously. Moreover, the recombinant Listeria vaccine vectors of the present invention may be administered in combination with e.g., serially, consecutively, or simultaneously, with other vaccine compositions. Non-limiting examples of such vaccine vectors are those for treating and/or prevention of any infection, disease or disorder caused by or related to a fungus, a parasite, a virus, or a bacterium; listeriosis, Ebola virus, SARS, small pox, hepatitis A, hepatitis B, hepatitis C, tuberculosis; diseases and disorders caused by human rhinovirus, HIV and AIDS, Herpes, polio, foot-and- mouth disease, rabies; diseases or disorders caused by or related to, rotavirus, influenza, coxsackie virus, human papilloma virus, SIN, malaria; cancer, e.g., tumors; and diseases or disorders caused by or related to infection by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, E. coli, flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites. A recombinant Listeria vaccine vector of the invention may be generated which encodes and expresses a heterologous antigen. The heterologous antigen, when expressed by L. innocua, is capable of providing protection in an animal against challenge by the infectious agent from which the heterologous antigen was derived, or which is capable of affecting tumor growth and metastasis in a manner which is of benefit to a host organism. Heterologous antigens which may be introduced into a recombinant Listeria vaccine vector by way of DNA encoding the same thus include any antigen which, when expressed by Listeria, serves to elicit an immune response which is of benefit to the host in which the response is induced, e.g., a human host. Heterologous antigens therefore include those specified by infectious agents, wherein an immune response directed against the antigen serves to prevent or treat disease caused by the agent. Such heterologous antigens include, but are not limited to, viral, bacterial, fungal or parasite proteins and any other proteins, glycoproteins, lipoprotein, glycolipids, and the like. Heterologous antigens also include those which provide benefit to a host organism which is at risk for acquiring or which is diagnosed as having a tumor. The host organism is preferably a mammal and most preferably, is a human. The term "heterologous antigen," as used herein, refers to a protein or peptide, a glycoprotein or glycopeptide, a lipoprotein or lipopeptide, or any other macromolecule which is not normally expressed in Listeria, which substantially conesponds to the same antigen in an infectious agent, a tumor cell or a tumor-related protein. For example, typical antigens may be classified as follows: protein antigens, such as ceruloplasmin and serum albumin; bacterial antigens, such as teichoic acids, flagellar antigens, capsular polysaccharides, and extra-cellular bacterial products and toxins; glycoproteins and glycolipids; viruses, such as animal, plant, and bacterial viruses; conjugated and synthetic antigens, such as proteinhapten conjugates, molecules expressed preferentially by tumors, versus normal tissue; synthetic polypeptides; and nucleic acids, such as ribonucleic acid and deoxyribonucleic acid. The term "infectious agent," as used herein, includes any agent which expresses an antigen which elicits a host cellular immune response. In one embodiment, the heterologous antigen is expressed by a recombinant Listeria vaccine vector, and is processed and presented to cytotoxic T cells upon infection of mammalian cells by the recombinant Listeria vaccine vector. The heterologous antigen expressed by Listeria species need not precisely match the corresponding unmodified antigen or protein in the tumor cell or infectious agent so long as it results in a T-cell response that recognizes the unmodified antigen or protein which is naturally expressed in the mammal. A heterologous antigen useful in vaccine development may be selected using knowledge available to the skilled artisan. For example, viral antigens which may be considered useful as heterologous antigens include, but are not limited to, the nucleoprotein (?NP) of influenza virus and the Gag proteins of HIV. Other heterologous antigens include, but are not limited to, HIV Env protein or its component parts gpl20 and gp41, HIV Nef protein, and the HIV Pol proteins, reverse transcriptase and protease. In addition, other viral antigens such as Ebola virus (EBON) antigens, such as, for example, EBON ?ΝP or glycoprotein (GP), either full-length or GP deleted in the mucin region of the molecule (Yang Z-Y, et al. (2000) Nat Med 6:886-9, 2000), small pox antigens, hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, Herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (F-VΪDN), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPN), for example the type 16 papilloma virus, the E7 protein thereof, and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIN) may be used. The heterologous antigens need not be limited to antigens of viral origin. Parasitic antigens, such as, for example, malarial antigens are included, as are fungal antigens, bacterial antigens and tumor antigens. Examples of antigens derived from bacteria are those derived from Bordetella pertussis (e.g., P69 protein and filamentous haemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E. coli antigens such as E. coli heat Labile toxin B subunit (LT-B), E. coli K88 antigens, and enterotoxigenic E. coli antigens. Other examples of antigens include Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens) and antigens of flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites, e.g., parasites of the genus plasmodium or babesia, for example Plasmodium falciparum, and peptides encoding immunogenic epitopes from the aforementioned antigens. By the term "tumor-related antigen," as used herein, is meant an antigen which affects tumor growth or metastasis in a host organism. The tumor-related antigen may be an antigen expressed by a tumor cell, or it may be an antigen which is expressed by a non-tumor cell, but which when so expressed, promotes the growth or metastasis of tumor cells. The types of tumor antigens and tumor-related antigens which may be introduced into Listeria by way of incorporating DΝA encoding the same, include any known or heretofore unknown tumor antigen, including, without limitation, the bcr/abl antigen in leukemia, HPNE6 and E7 antigens of the oncogenic virus associated with cervical cancer, the MAGE1 and MZ2-E antigens in or associated with melanoma, and the MNC-1 and HER-2 antigens in or associated with breast cancer. An infection, disease or disorder which may be treated or prevented by the administration of the recombinant Listeria vaccine vectors of the invention includes any infection, disease or disorder wherein a host immune response acts to prevent the infection, disease or disorder. Diseases, disorders, or infection which may be treated or prevented by the administration of the recombinant Listeria vaccine vectors of the invention include, but are not limited to, any infection, disease or disorder caused by or related to a fungus, parasite, virus, or bacteria, diseases, disorders or infections caused by or related to various agents used in biotereorism, listeriosis, Ebola virus, SARS, small pox, hepatitis A, hepatitis B, hepatitis C, diseases and disorders caused by human rhinovirus, HIN and AIDS, Herpes, polio, foot-and-mouth disease, rabies, diseases or disorders caused by or related to: rotavirus, influenza, coxsackie virus, human papilloma virus, SIN, malaria, cancer, e.g., tumors, and diseases or disorders caused by or related to infection by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, E. coli, flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites. The generation of recombinant Listeria vaccine vectors of the invention may be accomplished in a number of ways that are well known to those of skill in the art, including methods described in Lauer et al. ((2002) J. Bacteriology 184(15):4177-4186, the entire contents of which are incorporated herein by reference)). For example, shuttle integration vectors may be produced as described by Lauer et al. to integrate heterologous genes, e.g., hly, inlA, and/or prfA into Listeria, e.g., L. innocua. It will be appreciated that genes from L. innocua may also be removed, as long as the L. innocua retains its function, i.e., it is able to produce an immunogenic response in a subject. For example, the IplA gene is almost identical (97% homologous) between L. monocytogenes (gene number lmo0931) and L. innocua (gene number lin0931). In Listeria monocytogenes, this gene is important for growth and virulence. If it is deleted, the bacteria becomes less virulent (1/300 fold less virulent). Therefore, the IplA gene may be deleted from an L. innocua vaccine vector to decrease virulence. The vaccine vector of the invention may be, for example, lOkb, 20kb, 30kb, 40kb, or more in size. The introduction of DNA encoding a heterologous antigen into a strain of Listeria, e.g., L. innocua, to produce a recombinant Listeria vaccine vector of the invention may be accomplished, for example, by the creation of a recombinant Listeria in which DNA encoding the heterologous antigen is harbored on a vector, such as a plasmid for example, which plasmid is maintained and expressed in the Listeria species, e.g., a recombinant Listeria vector. Alternatively, DNA encoding the heterologous antigen may be stably integrated into the Listeria chromosome by employing, for example, transposon mutagenesis or by homologous recombination. A prefened method for producing recombinant Listeria having a gene encoding a heterologous antigen integrated into the chromosome thereof, is the induction of homologous recombination between a temperature-sensitive plasmid comprising DNA encoding the antigen and Listeria chromosomal DNA. Stable transformants of Listeria which express the desired antigen may be isolated and characterized as described herein in the experimental examples. This method of homologous recombination is advantageous in that site- directed insertion of DNA encoding the heterologous antigen is effected, thereby minimizing the possibility of disruption of other areas of the Listeria chromosome which may be essential for growth ofthis organism. Several approaches may be employed to express the heterologous antigen in Listeria species as will be understood by one skilled in the art once armed with the present disclosure. Genes encoding heterologous antigens are preferably designed to either facilitate secretion of the heterologous antigen from the bacterium or to facilitate expression of the heterologous antigen on the Listeria cell surface. Certain recombinant Listeria vaccine vectors of the invention may undergo osmotic lysis following infection of a host cell. Thus, if the Listeria which is introduced into the host cell comprises a vector, the vector is released into the cytoplasm of the host cell. The vector may comprise DNA encoding a heterologous antigen. Uptake of the vector DNA into the nucleus enables transcription of the DNA encoding the heterologous antigen and subsequent expression of the antigen in and/or secretion of the same from the infected host cell. Typically, the vector is a plasmid that is capable of replication in Listeria. The vector may encode a heterologous antigen, wherein expression of the antigen is under the control of eukaryotic promoter/regulatory sequences. Typical plasmids having suitable promoters that might be employed include, but are not limited to, pCMVbeta comprising the immediate early promoter/enhancer region of human cytomegalovirus, and those which include the SN40 early promoter region or the mouse mammary tumor virus LTR promoter region. It is also contemplated within the present invention that recombinant Listeria vaccine vectors may be employed for the purpose of stimulating a cytotoxic T cell (CTL) immune response against an infectious agent or a tumor cell, wherein the recombinant Listeria vaccine comprises a vector encoding a heterologous antigen that may be expressed using a eukaryotic expression system. According to the invention, the vector is propagated in the recombinant Listeria strain concomitant with the propagation of the recombinant Listeria strain itself. The vector may be, for example, a plasmid that is capable of replication in the recombinant Listeria vaccine vector may be lysogenic phage. The vector must contain a prokaryotic origin of replication and must not contain a eukaryotic origin of replication in order that the vector is capable of replication in a prokaryotic cell but, for safety reasons, is rendered incapable of replication in eukaryotic cells. A CTL immune response in a mammal is defined as the generation of cytotoxic

T cells capable of detectably lysing cells presenting an antigen against which the T-cell response is directed. Preferably, within the context of the present invention, the T cell response is directed against a heterologous antigen expressed in a recombinant Listeria vaccine or which is expressed by a vector which is delivered to a cell via Listeria infection. Assays for a cytotoxic T-cell response are well known in the art and include, for example, a chromium release assay (Frankel et al. (1995) J. Immunol. 155:4775- 4782). In addition to a chromium release assay, an assay for released lactic acid dehydrogenase may be performed using a Cytotox 96 kit obtained from Promega Biotech, WI. In prefened embodiments and using a chromium release assay, at an effector cell to target cell ratio of about 50:1, the percentage of target cell lysis is preferably at least about 10%ι above the background level of cell lysis. The background level of cell lysis is the percent lysis of cells which do not express the target antigen. More preferably, the percentage of target cell lysis is at least about 20% above background; more preferably, at least about 40% above background; more preferably, at least about 60% above background; and most preferably, at least about 70% above background. The preparation of vaccine compositions which contain recombinant Listeria as the active ingredient, is known to one skilled in the art. Recombinant Listeria vaccine vectors of the invention may be administered orally, pare terally, by injection, for example, either subcutaneously or intramuscularly, hi a prefened embodiment, the recombinant Listeria vaccine compositions of the invention are administered orally. While not intending to be bound by theory, it is believed that the route of administration of the recombinant Listeria vaccine vector may control whether the host response is a humoral immune response or a cellular immune response. Accordingly, in one embodiment, oral administration of the recombinant Listeria vaccine vector of the invention results in a cellular immune response. In another embodiment, oral administration of the recombinant Listeria vaccine vector, followed by an oral booster of the recombinant Listeria vaccine vector results in a cellular immune response. In another embodiment, oral administration of the recombinant Listeria vaccine vector, followed by an intramuscular booster of the recombinant Listeria vaccine vector results in a cellular and a humoral immune response Additional formulations which are suitable for other modes of admimstration include suppositories or formulations suitable for distribution as aerosols. The vaccine is advantageously presented in a lyopMlised form, for example in a capsular form, for oral administration to a patient. Such capsules may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", Cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose. These capsules may be used as such, or alternatively, the lyophilised material may be reconstituted prior to administration, e.g., as a suspension. Reconstitution is advantageously effected in buffer at a suitable pH to ensure the viability of the organisms. In order to protect the vaccine from gastric acidity, a sodium bicarbonate preparation is advantageously administered before each administration of the recombinant Listeria vaccine vector. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. The nucleic acid molecules or fusion polypeptides of the invention can be formulated into the vaccine or treatment compositions as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Vaccines maybe prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid, prior to infection can also be prepared. The active immunogenic ingredients are often mixed with carriers which are pharmaceutically acceptable and compatible with the active ingredient. The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in subjects to whom it is admimstered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, optionally, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, refened to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-( -2'-dipal mitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP) 19835 A, refened to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosporyl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA. In addition, immune modulating substances such as lymphokines (e.g., IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducers such as poly I:C can be used in combination with adjuvants described herein. Because Listeria is a gram-positive bacterium, it acts as a natural adjuvant, and therefore the addition of an adjuvant is not necessary. Both prophylactic and therapeutic recombinant Listeria vaccine vectors are contemplated as being within the scope of the present invention, that is, vaccines which are administered either prior to or subsequent to the onset of disease, and to those subjects which are at risk of acquiring the disease are included in the invention. The recombinant Listeria vaccine vectors of the invention may be directly administered to the subject, e.g., the human subject, without an adjuvant, i.e., because Listeria is a gram-positive bacteria, and therefore the addition of an adjuvant is not necessary. In one embodiment, the recombinant Listeria vaccine vectors of the invention may be administered to a subject orally, in a single dose or in multiple doses, as described herein. In another embodiment, the recombinant Listeria vaccine vectors of the invention maybe administered to a subject intramuscularly, in a single dose or in multiple doses, hi yet another embodiment, the recombinant Listeria vaccine vectors of the invention may be administered to a subject both orally and intramuscularly. In one embodiment, the recombinant Listeria vaccine vectors of the invention may be administered to a subject by a route determined by one of skillin the art in accordance with the type of infectious agent or tumor to be treated, as well as in accordance with the type of immunity that is desirable (see, e.g., Feltquate, et al. (1997) J. Immuno. 158:2278; McCluskie, et al. (1999) Mol. Med. 5:287; Bochner, et al. (1994) Ann. Rev. Immunol. 12:295; Weiner (1997) Immunol. Today 18:335; Kaufina m (1993) Ann. Rev. Immunol. 11:129). Furthermore, viability of the recombinant Listeria vaccine vectors of the invention does not depend on the administration of any secondary factor, in contrast to the attenuated L. monocytogenes vector (Lmdd), whose viability depends on the administration of exogenous D-alanine (as described in U.S. Patent ?No. 6,099,848). Vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g., the degree of protection or treatment desired. Suitable dosage ranges are of the order of 1 x 10⁴ to 3 x 10²⁰ colony forming units (CFUs) per vaccination with a preferred range from about 1 x 10⁵ to 3 x 10¹⁵ CFUs, and preferably in the range from about 1 x 10⁹to 3 x 10¹² CFUs. Suitable regiments for initial administration and boosters are also variable but are typified by an initial administration followed by subsequent oral administration, inoculations or other administrations. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of nucleic acid molecule or fusion polypeptides ofthis invention will depend, ter alia, upon the administration schedule, the unit dose of antigen administered, whether the nucleic acid molecule or fusion polypeptide is administered in combination with other therapeutic agents, and the immune status and health of the recipient. The vaccines can be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination can include 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. A booster inoculation following the initial inoculation, e.g., a booster administered orally following oral administration, may be used to induce an enhanced CTL response directed against the recombinant Listeria vaccine vector. Periodic boosters at intervals of 1-5 years, usually 3 years, are desirable to maintain the desired levels of protective immunity. The course of the immunization can be followed by in vitro proliferation assays of peripheral blood lymphocytes (PBLs) co-cultured with ESAT6 or ST-CF, and by measuring the levels of IFN-γ released from the primed lymphocytes. The assays can be performed using conventional labels, such as radionucleotides, enzymes, fluorescent labels and the like. These techniques are known to one skilled in the art and can be found in U.S. Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, which are hereby incorporated by reference. The phrase "in an amount and over a period of time effective to modulate an immune response to the antigen in the mammal" refers to a dosage and period of time in which modulation of an immune response in the recipient mammal or recipient subject occurs. In one embodiment, such an immune response can be observed when the recipient subject exhibits, for example, increased resistance to a challenge by the antigen against which the subject has been immunized using the recombinant Listeria vaccine vectors of the invention. The recombinant Listeria vaccine vectors of the invention are typically administered to the recipient animal or subject in the form of a vaccine composition by the routes and in the formulations described herein. In addition, the recombinant Listeria vaccine vectors of the invention can be administered in combination with other substances which influence immune responses including, but not limited to, cytokines, anaphylatoxins, cell-death inducing molecules, and cell surface molecules. The route of administration of the recombinant Listeria vaccine vectors may control whether the host response is a humoral immune response or a cellular immune response. In one embodiment, oral administration of the vaccine of the invention results in a cellular immune response. In another embodiment, oral administration of the vaccine followed by an oral booster of the vaccine of the invention results in a cellular immune response. . hi another embodiment, oral administration of the vaccine followed by an intramuscular booster of the vaccine of the invention results in a cellular and a humoral immune response. For treatment of cancer, the recombinant Listeria vaccine vectors of the invention may be used to protect individuals at high risk for cancer. In addition, the recombinant Listeria vaccine vectors may be used as an immunotherapeutic agent for the treatment of cancer following debulking of tumor growth by surgery, conventional chemotherapy, or radiation treatment. Patients receiving such treatment may be administered a recombinant Listeria vaccine vector which expresses a desired tumor antigen for the purpose of generating a CTL response against any residual tumor cells in the individual. The recombinant Listeria vaccine vectors of the present invention may also be used to inhibit the growth of any previously established tumors in a human by either eliciting a CTL response directed against the tumor cells per se, or by eliciting a CTL response against cells which synthesize tumor promoting factors, wherein such a CTL response serves to kill those cells thereby diminishing or ablating the growth of the tumor. The recombinant Listeria vaccine vectors of the invention may be maintained in storage until use. Storage may comprise freezing the recombinant Listeria vaccine vectors, or maintaining the vaccine at 4°C, room temperature, or the recombinant Listeria vaccine vectors may first be lyophilized and then stored. The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) an infection, disease or disorder characterized by the capability of the antigen which causes the disease or disorder to elicit a host cellular immune response. "Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent, e.g., a recombinant Listeria vaccine vector of the invention, optionally comprising a vector encoding a heterologous antigen, who has an infection, disease or disorder, a symptom of an infection, disease or disorder or a predisposition toward an infection, disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the infection, disease or disorder, the symptoms of the infection, disease or disorder, or the predisposition toward dise.ase. In one aspect, the invention provides a method for preventing in a subject, an infection, disease or disorder characterized by the capability of the antigen which causes the infection, disease or disorder to elicit a host cellular immune response, by administering to the subject a recombinant: Listeria vaccine vector of the invention, optionally encoding a heterologous antigen. Subjects at risk for a disease which is caused or contributed to infection by an antigen which is capable of eliciting a host cellular immune response, can be identified by, for example, any or a combination of diagnostic or prognostic assays known in the art or by assessment of risk for an individual or population for various risk factors including, for example, risk for biotenorism attack. Administration of a prophylactic agent of the invention can occur prior to the manifestation of symptoms c -aracteristic of the disease or disorder, or prior to exposure to the infectious agent, such ttiat an infection, disease or disorder is prevented or, alternatively, delayed in its progression.

This invention is further illustrated- by the following examples which should not be construed as limiting. The contents of -all references, patents, and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.

EXA-MPLES The following methodology descrfbed in the Materials and Methods section was used throughout the examples set forth below.

Materials and Methods Antigen-induced lymphocyte proliferation Peripheral blood mononuclear cells (P-V1BC) are isolated on Ficoll-hypaque gradients, washed and resuspended to a concentration of 5 x 10⁶/ml in RPMI 1640 medium (Sigma, St. Louis, MO) with glutamine plus 10% heat-inactivated fetal bovine serum and antibiotics. Cells are cultured in. 96-well microtiter plates at 37°C in an atmosphere of 8% CO₂. Cells are cultured for 96 hours in the presence of 10⁶-10⁸ heat- killed E. monocytogenes or 10-100 μg/ml recombinant LLO, the final 24 hours of which are in the presence of 1 μCi of ³H-thymidine. For ConA and PHA mitogen-induced proliferation, cells are cultured for 48 hours. Cells are harvested onto glass-fiber filters (Skatron, Sterling, VA) and counted in a liquid scintillation counter to measure ³H- thymidine incorporation. Results are expressed as cpm minus background counts of cells with no antigen or mitogen.

ELISPOT assays These assays are performed by the Immunology Core Laboratory at YNPRC or at DFCI using commercially available kits (BioSource International, hie, Camarillo, CA). For long-term ΕLISPOT assays, PBMC are restimulated with overlapping synthetic peptides (LLO or ΕBOV NC) in vitro for 7-10 days; for short-term ΕLISPOT assays, PBMC are restimulated with overlapping synthetic peptides in vitro for 4 hours.

Mouse CTL assays Effector cells are splenic mononuclear cells, which are isolated from vaccinated or control mice and restimulated (2xl0⁶/ml) in vitro with 1 μM EBOV GP peptide (TELRTFSI), an identified CTL epitope in H-2^kmice), NP peptide (VYQVNNLEEIC), an identified CTL epitope in H-2^k mice) or LLO peptide 91-99 (containing a CTL epitope of H-2^d mice) for 7-10 days. Alternatively, effector cells are restimulated in vitro with 1 μM overlapping synthetic peptides conesponding to the inserts (LLO or EBOV NC protein; EBOV NC peptides are purchased from Sigma Genosys, Woodlands, TX) for 7-10 days. Target cells are P815 cells (H-2^d, for BALB/c mice) or EL-4 cells (H-2^b, for C57BL/6 mice). These cells are labeled with ⁵¹Cr (70 μCi/2 x 10⁶ cells; PerkinElmer, Boston, ?MA) and pulsed for 1 hour with or without overlapping synthetic peptides conesponding to the insert proteins or the vector proteins (1 μM), or infected 6 hours to overnight with vaccinia virus [2 plaque forming units (pfu)/target cell] expressing the insert protein or the protein of the vector itself or wild-type vaccinia virus (obtained from Therion, Cambridge, MA). Effector cells and target cells are co-cultured at different ratios for 6 hours and cytolysis is determined by ⁵¹Cr release from target cells. The percentage specific ⁵¹Cr release is calculated as: 100 (experimental release - spontaneous release)/ (maximum release - spontaneous release). Maximum release is determined from supernatants of cells that are lysedby the addition of 5% Triton-X 100. Spontaneous release is determined from target cells incn-bated without the addition of effector cells.

Monkey CTL assays Effector cells (PBMC) isolated from vaccinated or control monkeys, are isolated from anti-coagulated blood by Ficoll-Hypaque centrifugation or by centrifugation in heparin CRT tubes (Becton-Dickson, Franklin Lakes, NJ) and restimulated with stimulators [herpes virus papio-transformed autologous B-cell lines (BLCL) transfected with vaccinia virus expressing LLO, EBOV GP or EBO NC] for 7 -10 days. Alternatively, effector cells are stimulated with overlapping synthetic peptides conesponding to the inserts (LLO or EBOV NC protein) for 7 - 10 days. Target cells are BLCL transfected overnight with vaccinia virus expressing LLO, EBOV GP or EBOV NC, or BLCL pulsed overnight with overlapping synthetic peptides conesponding to the insert proteins or the vector proteins. Effector cells and target cells are cocultured at different ratios for 6 hours and cytolysis is determined by ⁵¹Cr release from target cells. The percentage specific ⁵¹Cr release is calculated as: 100 (experimental release - spontaneous release)/(maximum release - spontaneous release). Maximum release is determined from supernatants of cells that are lysed by the addition of 5% Triton-X 100. Spontaneous release is determined from the target cells incubated without the addition of effector cells.

Real-time quantitative DNA PCR for L. monocytogenes or L. innocua To measure bacteremia with either L. monocytogenes or L. innocua-based organisms, a real-time quantitative PCR assay published by Hein et αl. (2001) Res. Microbiol. 152(1):137, is utilized. The probe recognizes the iαp gene present in both species; the assay yields a fragment of 175bp for L. monocytogenes and 309 bp forE. innocua, respectively. Out of 42 different L. monocytogenes strains and 33 different L. innocua strains tested, the PCR assay yielded positive results for all strains; as few as 6 copies of the iap gene per PCR were detectable. This test was significantly more rapid compared to the standard plate count method to detect and quantify L. monocytogenes or E. innocua in milk, and real-time quantitative PCR yielded 1 to 2 logs higher iap gene copy numbers compared to cfu obtained by the plate count method. Example 1 ; Construction of L. innocua-hly-inlA, which expresses LLO and internalin A. This example describes the construction of anZ. innocua vector which contains the hly gene and the inlA gene. A shuttle vector between E. coli and Listeria axe constructed according to Shen H, et αl. (1995) Proc NαtlAcαdSci USA 89:7581-5. A 5.7 kb DNA fragment covering the region from gcαD to Idh (1369-7097 in the L. innocua genome, segment 2/12) is amplified from the L. innocua chromosomal DNA. This fragment is ligated with pBR322 (or pUC18) and pE194 to form the shuttle vector. Then, the 1.6 kb hly and 2.4 kb inlA (including promoter and coding sequences) are amplified from the L. monocytogenes chromosomal DNA, respectively, and inserted between prs and orfZ (Figure 1). The shuttle vector containing hly and inlA axe transfected into L. innocua by electroporation. After a double allelic exchange recombination reaction, the genes of interest, hly and inlA, are left inserted in the bacterial chromosome between genes gcaD and Idh, whereas the remainder of the shuttle vector is lost from the chromosome.

Example 2: Construction of X. innocua-prfA-hly-inlA to express PrfA, LLO and internalin A This example describes the constructions of anJ. innocua vector containing the prf gene, the inlA gene, and the inlA gene. To add prfA into the shuttle vector constructed as described above in Example 1, the 4.1 kb fragment containing prfA-plcA-hly is amplified from L. monocytogenes chromosomal DNA and replaces hly (Figure 1). The resulting shuttle vector containing prfA, hly and inlA will be transfected into L. innocua by electroporation and the recombinant L. innocua and screened thereafter.

Example 3: Development of an optimal live Listeria vector as an oral vaccine . In this Example, vectors produced as described in Examples 1 and 2 are evaluated for safety, immunogenicity, and efficacy for the development of an optimal live Listeria vector for use as an oral vaccine.

Safety, Immunogenicity, and Efficacy Testing in Mice In this experiment, the vectors produced as described above are tested for their ability to cause morbidity in mice. Classical determinations of 50% lethal doses (LD₅₀) are used. Mice each receive increasing doses of the candidate vaccine vectors. BALB/c mice are used, in which the LD₅₀ for wild-type L. monocytogenes is 10⁴ organisms (Bany RA, et al. (1992) Infect Immun 60:1625-32). The mice are followed prospectively for signs of illness and examined daily. Every third day, 50 ul of blood is collected form the retro-orbital sinus to measure bacteremia by real-time DNA PCR. Wild-type L. monocytogenes organisms survive and replicate in the spleens and livers of infected BALB/c mice for up to seven days, with peak bacterial replication occurring on days 2 or 3 post-challenge. The recombinant L. innocua vaccine vector has been designed to be taken up by cells in the gastrointestinal tract and to enter the host cell cytosol after escaping phagosomes. Therefore, the vector will induce protective cellular immune responses against oral challenge from wild-type L. monocytogenes.

Safety, Immunogenicity, and Efficacy Testing in Juvenile Macques A safe, immunogenic dose of the recombinant Listeria vaccine vector is subsequently identified in experiments using juvenile monkeys. Four groups of four animals are enrolled. The control group is left untreated, whereas the other three groups receive different oral doses of the recombinant Listeria vaccine vector. The animals are followed prospectively for development of specific anti-E. monocytogenes immune responses. If necessary, oral boosts are given. Challenge with wild-type L. monocytogenes is performed only if specific anti- L. monocytogenes cellular immune responses develop. Post-challenge, all monkeys are examined daily for signs of bacteremia and clinical disease. Blood and stools are also monitored. Strong cellular immune responses at the level of mucosal tissues and the systemic circulation provide a high degree of protection against oral L. monocytogenes challenge. In contrast, naive control monkeys are bacteremic and show signs of listeriosis. Real-time PCR allows the definite diagnosis to be made within a short period of time.

Safety, Immunogenicity, and Efficacy) in Pregnant Rhesus Monkeys The dose of the recombinant Listeria vaccine vector found to be safe and immunogenic in juvenile monkeys is well-tolerated when given to pregnant dams during the third trimester. In order to test this hypothesis, two groups of four pregnant animals are utilized. The control group is left untreated, and group 2 receives the attenuated Listeria vaccine of the invention at the optimal dose as detennined in juvenile macaques. Because fetal loss is most pronounced when rhesus monkey dams are infected with wild- type E. monocytogenes at the beginning of the third trimester, the first vaccimation is given between gestational days 110 and 115 (gestation in rhesus monkeys is 165 days). The pregnant dams are closely monitored for signs of septicemia, and shedd-ing of viable vaccine organism in the stool is measured. Newborn infants or aborted fetuses are tested for signs of listeriosis. Oral challenge of the adult females with wild-type L. monocytogenes is postponed for several months. This delay allows monitoring of the animals over an extended period of time for signs of vaccine pathogenicity. At the same time, the delayed challenge permits investigation into whether the antiviral immunity induced by the attenuated Listeria vaccine is long-lived. Without giving any intervening boosts, the animals -are challenged orally at a late time point. This mimics the situation of human vaccine recipients who may be exposed to L. monocytogenes -contaminated food long after their peak immunity from the last boost has waned. The oral challenge with L. monocytogenes in the previous experiment is conducted two weeks after the last boost, i.e., at the time when anamnestic immune responses peak and make resistance to the pathogenic cliallenge organism likely.

Safety, Immunogenicity, and Efficacy in Neonatal Monkeys Neonatal rhesus monkeys are more susceptible to L. monocytogenes -induced disease than adults and represent the most sensitive hosts to reveal any residταal pathogenicity of the recombinant Listeria vaccine vector. Safe, immunogendc doses can be found that will protect infant macaques after vaccination in the newborn period against subsequent challenge with wild-type L. monocytogenes. Two groups of four rhesus monkey neonates are enrolled. One group will serve as naive controls, and the other group is vaccinated orally with the live attenruated Listeria vector studied previously in juvenile and pregnant macaques. The v^accine dose is adjusted according to body weight. The infants are monitored closely for signs of septicemia. Immunogenicity are assessed as outlined above. If needed, ora-1 boosts are given. Two weeks after the last boost, animals in both groups are challenged with wild- type L. monocytogenes (the dose will be adjusted according to body weight). Post- challenge, the infants will be monitored for signs of listeriosis. The experimental protocol and methods are as follows. The animals are born in the breeding conals by normal spontaneous vaginal delivery. After birth, they are separated from their mothers and brought to the biocontainment suite, where they are hand-reared (captive rhesus monkey females often neglect their offspring; thus, hand- rearing is the most reliable way to conduct vaccine studies in newborns). Because of the small size of the infants (500 grams at birth), blood drawing is limited. Whole blood is collected in pediatric tubes. The scaled-down assays for neonatal immunization studies are used. Stool samples are also collected to evaluate shedding of viable Listeria organisms. Based upon the precedent set by oral vaccination of human neonates performed in the late 1950's with live attenuated polio virus vaccines (Sabin AB, et al. (1963b) Pediatrics 31:641-50; Wanen RJ, et al. (1964) Pediatrics 34:4-13)), it is not anticipated that the neonatal encounter with the live attenuated Listeria vaccine vector will induce tolerance. These infants were vaccinated orally during the first months of life. To test whether they had generated immunity, they were "challenged" a few months later with the live attenuated oral vaccine again, and stool samples were examined for excretion of live virus. If the vaccinated infants had anti-polio virus immunity, replication of the vaccine strain upon re-exposure would be drastically decreased or prevented completely compared to naϊve children. Indeed, this was the case. None of the children vaccinated as neonates were reported to develop poliomyelitis, thus indicating that the live attenuated vaccine was safe and immunogenic in these young infants. Likewise, evidence of tolerization was found no when neonatal rhesus monkeys were vaccinated with the DNA prime/protein boost regimen ( Rasmussen RA, et al. (2002a) JMed Primatol 31:40-60). Furthermore, exposure of newborn mice to a live attenuated L. monocytogenes vector encoding HIV Gag (Lmdd-gag) whose viability depends on exogenous D-alanine, did not result in tolerization, but rather in long-term protection when these animals were challenged several months later (Rayevskaya M, et al. (2002) J Virol 76:918-22). Example 4: Development of an optimal live Listeria vector as oral vaccine against EBOV Based on the biology of live Listeria vectors, a foreign gene encoded by a live Listeria vector is targeted to antigen presenting cells (APC) and presented effectively to the MHC class I antigen presentation pathway. Potent CTL is induced against the EBOV genes encoded by the candidate live attenuated Listeria vector. A single vaccination may induce protection, as judged from previous studies in mouse models involving Listeria vectors with gene inserts from different pathogens. A unique feature of the vaccine vector of the invention is oral delivery. A live attenuated Listeria vector with the desired characteristics, namely safety and immunogenicity after oral vaccination, even in sensitive pregnant and neonatal animal hosts is identified as described above. Different Listeria-EBOV vectors are generated, one encoding the EBOV NP gene. A VEE vector encoding this EBOV antigen induced CTL in mice that protected naϊve recipients upon adoptive transfer (Wilson JA and Hart MK (2001) J Virol 75 :2660-4). The EBOV GP antigen is also cloned into the attenuated Listeria vector; however, because this gene product alone has shown cytopathicity to cultured cells and to aortic explants in organ culture, the mucin- like region that has been implicated in this endothelial cytopathicity is deleted. Vectors encoding the mutated GP genes of various EBOV strains may be generated. Initial safety and immunogenicity studies of such recombinant Listeria-EBO V vectors are carried out in mice. If the induction of potent cellular immune responses is displayed in mice, challenge with pathogenic EBOV follows. After testing the safety, immunity and efficacy of five different attenuated Listeria vectors in mice, the one with the best safety/efficacy profile against wild-type L. monocytogenes challenge in mice is selected to construct shuttle vectors to express EBOV nucleocapsid-associated nucleoprotein (NP) or the surface transmembrane glycoproteins (GP) after deleting the serine-threonine-rich, mucin-like-region (Xu L, et al. (1998) NatMed 4:37-42; Sullivan NJ, et al. (2000) Nature 408:605-9; Yang Z-Y, et al. (2000) Nat Med 6:886-9, 2000). EBOV antigens are expressed using the hly promoter. The genes encoding the following antigens are PCR amplified and cloned into the selected shuttle vectors: a. L-NP(Z), expressing NP of EBOV (strain Zaire) b. L-GP-Δmuc(Z), expressing GP of EBOV (strain Zaire) c. L-GP-Δmuc(S), expressing GP of EBOV (strain Sudan) d. L-GP-Δmuc(IC), expressing GP of EBOV (strain Ivory Coast) e. L-GP-Δmuc(R), expressing GP of EBOV (strain Reston)

To construct attenuated Listeria vectors of the invention expressing EBOV antigens, the plasmid pJJD180 is linearized with EcoRl and Hindlll to insert the EBOV ? P and GP antigen gene fragments. The Zaire EBOV nucleoprotein gene is 2.2 kb and is amplified by PCR to include EcoRl and Hindlll recognition sites in the 5' and 3' ends, respectively. After this fragment is PCR-cloned and confirmed by DNA sequence analysis, it is subcloned into pJJD180 between EcoRl and Hindlll sites, so that the expression of ?NP(Z) will be regulated by the hly promoter. GP(Z) is amplified as two different fragments (5 '-end and 3 '-end) without the internal mucin-like region (amino acids 315-505), then the two fragments are ligated together and inserted between EcoRlll and Hindlll sites of pJJDl 80. Other vectors expressing the GP(Δmuc) from different ΕBOV strains of Sudan, Ivory cost and Reston isolates may be constructed in the same way. The expression vectors are then transfected into the attenuated Listeria vectors of the invention to generate recombinant ΕBOV LO vaccines.

Safety Testing The LD₅₀ of each of the newly created Listeria-EBOV vectors in mice is tested. The ΕBOV inserts will not significantly alter the LD₅₀ of the attenuated Listeria vector itself. This first safety evaluation is conducted in adult mice. If the LD₅₀ values of the recombinant vectors encoding ΕBOV genes are in the same order of magnitude as that of the vector itself, the Listeria-EBOV ^'vectors are tested in neonatal mice by performing an LD₅₀ assessment.

Immunogenicity Groups of mice are vaccinated with the recombinant Listeria-EBOV vectors. One group of control animals receives the empty vector, and the second control group is left naive. If necessary, the mice are boosted. The animals are followed prospectively for the development of cellular and humoral immunity to the vector inserts and to the vector itself. The mice generate potent CTL responses against the EBOV antigens encoded by the various vectors. Safety and immunogenicity are tested after oral vaccination with live attenuated Listeria-EBOV vectors in rhesus monkeys using a multigenic approach by mixing vectors expressing either ?NP or mutated GP derived from different virus strains. Four groups of four rhesus monkeys are used to evaluate safety and immunogenicity of the Listeria-EBOV vector. Group 1 are vaccinated with Listeria vector encoding the EBOV ?NP antigen; group 2 receive the Listeria vector encoding the mutated GP of the Zaire strain; and group 3 are vaccinated with both vectors at the same time. Group 4, the control group, receive the empty vector only. All animals are followed prospectively for the development of immune responses to the vector and to the different inserts. If potent, specific immune responses are seen. The challenge is performed with homologous EBOV (strain Zaire). Vaccination against two EBOV genes may provide better protection than vaccination against only one viral antigen. Furthermore, vaccination of rhesus monkeys with attenuated Listeria vectors encoding the ?NP antigen and mucin-deleted GP genes of multiple different strains may induce the same or increased potent cellular immune responses as vaccination with GP from only one strain.

Example 5: Oral priming followed by oral or intramuscular boosting with recombinant Listeria of the invention The route of administration of the vaccine of the invention may determine the immune response by the host organism, e.g., a cellular immune response versus a humoral immune response. To test the immune response of the vaccines of the invention, juvenile rhesus monkeys of Chinese origin are vaccinated with recombinant L. innocua vaccine alone or encoding a specific antigen, e.g., ?NP or GP of EBOV. One group of monkeys receive only oral vaccine at weeks 0, 6, and 19. In contrast, a second group of monkeys receives 2 oral doses of the vaccine, followed by an intramuscular does at week 19. Anti-vector immune responses are assessed by performing proliferation and ELISA assays. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is Claimed:

1. A vaccine comprising a recombinant Listeria innocua strain, wherein said recombinant Listeria innocua strain comprises one or more heterologous genes, and wherein said recombinant Listeria innocua strain is capable of eliciting an immunogenic response in a subject.

2. The recombinant Listeria innocua strain of claim 1, wherein said heterologous gene(s) are derived from Listeria monocytogenes.

3. The recombinant Listeria innocua strain of claim 1, wherein said heterologous gene is the hly gene.

4. The recombinant Listeria innocua strain of claim 1, wherein said heterologous genes are the hly gene the inlA gene.

5. The recombinant Listeria innocua strain of claim 1, wherein said heterologous genes are the hly gene, the inlA gene, and a gene encoding a transcription factor.

6. The recombinant Listeria innocua strain of claim 5, wherein said transcription factor is the prfA gene.

7. The recombinant Listeria innocua strain of claim 1, wherein administering said recombinant Listeria innocua strain causes the production of a cytotoxic T-cell response in a subject.

8. The recombinant Listeria innocua strain of claim 1, furthering comprising DNA encoding one or more heterologous antigens.

9. The recombinant Listeria innocua strain of claim 1, further comprising a vector comprising a DNA encoding one or more heterologous antigens.

10. The recombinant Listeria innocua strain of claim 8 or 9, wherein said antigen is selected from the group consisting of: a viral protein, a bacterial protein, a fungal protein, a surface protein, a glycoprotein, a lipoprotein, or a glycolipid.

11. The recombinant Listeria innocua strain of claim 8 or 9, wherein the foreign antigen is selected from the group consisting of: Ebola virus, HIV, SARS, a small pox antigen, hepatitis A, B or C virus, human rhmovirus, Herpes simplex virus, poliovirus (type 2 or type 3), foot-and-mouth disease virus (F?MDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), the E7 protein of HPV, and fragments containing the E7 protein or its epitopes, simian immunodeficiency virus (SIV), malarial antigens, fungal antigens, bacterial antigens, tumor antigens, antigens derived from Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, and E. coli antigens, Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens), and antigens of parasites including flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria.

12. A method of eliciting a cytotoxic T-cell response to an antigen of an infectious agent in a subject comprising administering to said subject an effective amount of a vaccine comprising recombinant Listeria innocua, wherein said recombinant Listeria innocua comprises one or more heterologous genes.

13. The method of claim 12, wherein said heterologous gene(s) are derived from Listeria monocytogenes.

14. The method of claim 12, wherein said heterologous gene is the hly gene.

15. The method of claim 12, wherein said heterologous genes are the hly gene and the inlA gene.

16. The method of claim 12, wherein said heterologous genes are the hly gene, the inlA gene, and a gene encoding a transcription factor.

17. The method of claim 12, wherein said transcription factor is the prfA gene.

18. The method of claim 12, wherein said subject is a human.

19. The method of claim 12, wherein said vaccine is administered orally.

20. The method of claim 12, wherein said vaccine is administered in multiple doses.

21. The method of claim 12, wherein said recombinant Listeria innocua further comprises DNA encoding a heterologous antigen.

22. The method of claim 12, wherein said recombinant Listeria innocua further comprises a vector comprising a DNA encoding a heterologous antigen.

23. The method of claim 21 or 22, wherein said antigen is selected from the group consisting of: a viral protein, a bacterial protein, a fungal protein, a parasite protein, a glycoprotein, a lipoprotein, or a glycolipid.

24. The method of claim 21 or 22, wherein the heterologous antigen is selected from the group consisting of: Ebola virus, HIV, SARS, a small pox antigen, hepatitis A, B or C virus, human rhinovirus, Herpes simplex virus, poliovirus (type 2 or type 3), foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), the E7 protein of HPV, and fragments containing the E7 protein or its epitopes, simian immunodeficiency virus (SIV), malarial antigens, fungal antigens, bacterial antigens, tumor antigens, antigens derived from Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, andE. coli antigens, Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens), and antigens of parasites including flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria..

25. A method of treating or preventing infection, disease or disorder in a subject comprising administering to said subject a vaccine comprising a recombinant Listeria innocua strain, wherein said recombinant Listeria strain comprises one or more heterologous genes, thereby treating or preventing infection, disease or disorder in said subject.

26. The method of claim 25, wherein said subject is a human.

27. The method of claim 25, wherein said vaccine is administered orally.

28. The method of claim 27, wherein said vaccine is admimstered in multiple doses.

29. The method of claim 25, wherein said infection, disease or disorder is selected from the group consisting of: any infection, disease or disorder caused by or related to a fungus, parasite, virus, or bacteria, listeriosis, Ebola virus, SARS, small pox, hepatitis A, hepatitis B, hepatitis C, diseases and disorders caused by human rhinovirus, HIV and AIDS, Herpes, polio, foot-and-mouth disease, rabies, diseases or disorders caused by or related to: rotavirus, influenza, coxsackie vims, human papilloma virus, SIV, malaria, cancer, and diseases or disorders caused by or related to infection by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, E. coli, flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites.