Self-destructing and tumor-navigating bacterial host-vector systems

Abstract

The present disclosure provides genetically modified Salmonella (GMS) strains with enhanced self-destructing and tumor-navigating activity.

Description

SELF-DESTRUCTING AND TUMOR-NAVIGATING BACTERIAL HOST-VECTOR SYSTEMS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 697,441 filed on September 20, 2024, the content of which is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R21 CA249517 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The content of the electronic sequence listing (11262401536.xml; Size 15,517 bytes; and Date of Creation: September 22, 2025) is herein incorporated by reference in its entirety. BACKGROUND

[0004] Recombinant microorganisms play a crucial role in various applications, including as live vaccines designed to elicit an immune response. When using a recombinant microorganism as a live vaccine for vertebrates, the microorganism must be free of antibiotics and known antibiotic resistance genes, particularly those associated with mobile genetic elements such as plasmids or transposons. It must be able to adhere to, invade, and persist in the lymphoid tissues of the vertebrate. It should continually expose these immune sites to the antigen to stimulate a robust and sustained immune response. Attenuated pathogens are particularly beneficial because they have developed sophisticated mechanisms to access lymphoid tissues, ensuring effective interaction with the immune system. For a live recombinant vaccine to be effective, it must be properly attenuated and safely contained. There is a need for a recombinant bacterium that can serve as a live vaccine with features including attenuation, biocontainment, antigen expression or synthesis, and the ability to deliver antigen or nucleic acid encoding an antigen to host cells.SUMMARY OF THE DISCLOSURE

[0005] In an aspect, provided herein is a genetically modified Salmonella bacterium comprising a ΔPmurA::TT araC PBADmurA mutation; and a ΔasdA::TT araC PBADc2 mutation, optionally comprising a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 1. In embodiments, the bacterium further comprises mutations: Δ(gmd-fcl); ΔrelA; and ΔendA. In embodiments, the bacterium further comprises mutations: ΔaraBAD; Δpmi; ΔrelA::araC PBAD lacI; and Δgmd-fcl ΔrelA::araC PBADlacI. In embodiments, the bacterium further comprises mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM) ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp IpxE; and ΔendA. In embodiments, the bacterium further comprises mutations: Δ(araC PBAD)::P22 PRaraBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBADlacI TT; ΔpagP::PlpplpxE; and ΔendA. In embodiments, the bacterium further comprises mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza- wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; ΔendA; and Δ Ptar::PtrcΔlacOtar. In embodiments, the bacterium further comprises mutations: Δ(araC PBAD)::P22 PRaraBAD; Δ(wza- wcaM); Δpmi; ΔrelA; ΔpagP::PlpplpxE; ΔendA; ΔPtar::PtrcΔlacOtar; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; ΔendA; ΔPtar::PtrcΔlacOtar; ΔPtsr::Ptrc ΔlacO tsr. In embodiments, the bacterium further comprises mutations: Δ(araC PBAD)::P22 PRaraBAD; Δ(wza- wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; ΔendA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza- wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::Ptrc ΔlacO tar; ΔPtsr::Ptrc ΔlacO tsr; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::PlpplpxE; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::Ptrc ΔlacOtar; ΔPtsr::PtrcΔlacO tsr; and Δtrg.

[0006] In an aspect, provided herein is a genetically modified Salmonella bacterium comprising a ΔPmurA::TT araC PBADmurA mutation; and a ΔasdA::TT araC PBADc2 mutation, optionally comprising a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 2. In embodiments, the bacterium further comprises mutations: ΔaraBAD; ΔaraE; Δ(gmd-fcl); ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; andΔsseL. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza- wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacO hilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg. In embodiments, the bacterium further comprises mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::PlpplpxE; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

[0007] The bacterium may further comprise a recombinant polynucleotide encoding at least one of an antigen and an effector protein.

[0008] In another aspect, provided herein is a method of treating a subject in need thereof, the method comprising administering any of the genetically modified Salmonella bacteria described herein. DETAILED DESCRIPTION

[0009] The inventors have developed self-destructing and self-destructing tumor-navigating genetically modified Salmonella (GMS) host-vector systems as protein, nucleic acid vaccine vector and therapeutic delivery platforms. These GMS strains may be used to induce mucosal, systemic, and cellular immune responses in mice against viral, bacterial, and parasite pathogens ortarget the tumor microenvironment (TME), deliver anti-cancer materials, and inhibit tumor growth and cancer metastases.

[0010] The vector systems described herein include mutations of the inventors’ previously developed GMS strains, which feature regulated delayed lysis and can serve as a vaccine or therapeutic DNA / RNA / protein delivery platform. The Salmonella lysis strains harbor a deletion of asdA and the arabinose-regulated expression of murA, two genes required for the synthesis of the peptidoglycan layer of the bacterial cell wall. They also contain additional mutations intended to enhance bacterial cell lysis, and antigen delivery. The lysis vector cooperatively works with its host Salmonella lysis strain to facilitate the arabinose-dependent bacterial cell wall synthesis needed for bacterial reproduction. Upon invasion of host tissues, which is an arabinose-free environment, the synthesis of the bacterial cell wall eventually ceases. This results in bacterial cell lysis, release of bacterial cell content (e.g. therapeutic proteins or DNA expression vectors) after bacteria accumulates in host tissues and finally accomplishes Salmonella self-eradicating, contributing to the improved efficacy.

[0011] To further improve the safety features of Salmonella and enable systemic therapeutic interventions the Lipid A component, which is responsible for the toxicity of lipopolysaccharide (LPS) was modified. LpxE, an inner membrane phosphatase from Francisella tularensis can selectively remove the 1-phosphate group of lipid A in living cells of Salmonella to generate a close analog of monophosphoryl lipid A (MPLA). TLR4 is required for protective immune response and killing of cancer cells by inducing an efficient cancer antigen-specific cytotoxic T cell immune response. As a TLR4 agonist and adjuvant, MPLA improves vaccine efficacy, induces dendritic cell maturation, primarily induces a Th1 response, indirectly reduces the threshold for activation of Th1 cells, and upregulates MHC class II molecules. Therefore, a LpxE-expressing cassette was introduced into GMS, which maintains or enhances immunostimulatory benefits but possesses reduced toxicity. Besides, Salmonella processes multiple natural killer (NK) cell stimulators, including flagellin. Flagellin has been shown to directly cause NK cell proliferation as well as activation through dendritic cells (DCs). These self-destructing GMS strains induce mucosal, systemic, and cellular immune responses in mice against viral, bacterial, and parasite pathogens.

[0012] These self-destructing GMS are also modified for specific anti-cancer material delivery. Salmonella has also been extensively explored as a potential cancer treatment. Many tumorsrelease small molecules that attract Salmonella to the tumors. Specifically, the aspartate chemoreceptor (Tar) initiates chemotaxis toward tumor cylindroids, the serine chemoreceptor (Tsr) initiates cylindroid penetration, and the ribose chemoreceptor (Trg) directs Salmonella toward the necrotic area. To transform self-destructing Salmonella into a universal tumor-targeting delivery vehicle for cancer therapy, the inventors have previously reprogrammed the chemotaxis system resulting in tumor-navigating GMS strains for protein delivery and nucleic acid vaccine vector delivery.

[0013] The inventors sought to improve and optimize these self-destructing and tumor-navigating bacteria strains to harbor plasmids and serve as universal improved and optimized host-vector systems for protein, nucleic acid vaccine vector and therapeutic delivery. Provided are universal self-destructing and tumor-navigating GMS host-vector systems as protein, nucleic acid vaccine vector and therapeutic delivery platforms. These GMS strains may be used to induce mucosal, systemic, and cellular immune responses in mice against viral, bacterial, and parasite pathogens and target TME, release anti-tumor materials, and inhibit tumor growth and cancer metastasis.

[0014] I. Vector

[0015] A recombinant bacterium capable of the regulated expression of at least one nucleic acid sequence encoding an antigen or effector protein comprises, in part, a vector. The vector comprises a nucleic acid sequence encoding at least one antigen or effector protein of interest operably linked to a promoter. The promoter is regulated by the chromosomally encoded repressor, such that the expression of the nucleic acid sequence encoding an antigen or effector protein of interest is repressed during in vitro growth of the bacterium, but the bacterium is capable of high-level synthesis of the antigen or effector protein in an animal or human host. In certain embodiments, however, the promoter may also be regulated by a plasmid encoded repressor.

[0016] As used herein, “vector” refers to an autonomously replicating nucleic acid unit. A “vector” encompasses any known type of vector, including viral, cosmid, phasmid, and plasmid vectors. The most preferred type of vector is a plasmid vector. As is well known in the art, plasmids and other vectors may possess a wide array of promoters, multiple cloning sequences, transcription terminators, etc., and vectors may be selected so as to control the level of expression of the nucleic acid sequence encoding an antigen by controlling the relative copy number of the vector. In some instances, in which the vector might encode a surface localized adhesin as the antigen, or an antigen capable of stimulating T-cell immunity, it may be preferable to use a vector with a low copynumber such as at least two, three, four, five, six, seven, eight, nine, or ten copies per bacterial cell. A non-limiting example of a low copy number vector may be a vector comprising the pSC101 ori. In other cases, an intermediate copy number vector might be optimal for inducing desired immune responses. For instance, an intermediate copy number vector may have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell. A non-limiting example of an intermediate copy number vector may be a vector comprising the p15 A ori.

[0017] In still other cases, a high copy number vector might be optimal for the induction of maximal antibody responses. A high copy number vector may have at least 31, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies per bacterial cell. In some embodiments, a high copy number vector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 copies per bacterial cell. Non-limiting examples of high copy number vectors may include a vector comprising the pBR ori or the pUC ori.

[0018] Additionally, vector copy number may be increased by selecting for mutations that increase plasmid copy number. These mutations may occur in the bacterial chromosome but are more likely to occur in the plasmid vector.

[0019] Preferably, vectors used herein do not comprise antibiotic resistance markers to select for maintenance of the vector.

[0020] A. Lysis Vector

[0021] Combining the asdA and murA systems provides redundant mechanisms to ensure cell death. First, it is needed to reduce the amount of Asd produced from our plasmid. The araC PBAD promoter-activator was derived from an Escherichia coli B / r strain. When substituted the araC PBADpromoter-activator from E. coli K-12 strain ^289, transcription from the plasmid was more tightly regulated and arabinose-dependent growth was achieved. Then a murA gene was inserted in between the PBAD promoter and the asdA gene to further decrease the transcription level of asdA. Finally, P22 PR, a C2-regulated promoter, was introduced with opposite polarity at the 3 ends of the asd gene to interfere with transcription of the plasmid asdA and murA genes and to direct synthesis of antisense mRNA to block translation of mRNA transcribed from these genes during programmed lysis when arabinose is absent. Transcription terminators flank all plasmid domains so that expression in one domain does not affect the transcriptional activities of any other domain. The resulting plasmid was designated pYA3681 (SEQ ID NO: 1. The plasmid comprises asequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. The bacterium may comprise this plasmid.

[0022] In embodiments, thebacterium comprises a lysis vector comprising a sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2 (pYA4545).

[0023] 1. Antigen or Effector Protein

[0024] As used herein, “antigen” refers to a biomolecule capable of eliciting an immune response in a host. In some embodiments, an antigen may be a protein, or fragment of a protein, or a nucleic acid. In an exemplary embodiment, the antigen elicits a protective immune response. As used herein, “protective” means that the immune response decreases the size of a tumor, decreases metastases, and / or contributes to the lessening of any symptoms associated with a tumor. The use of the term “protective” does not necessarily require that the host is completely protected from the effects of the tumor. As used herein, “effector protein” refers to a biomolecule capable of inhibiting tumor cell growth. In some embodiments, an effector protein may induce programmed cell death (e.g. apoptosis or pyroptosis) in tumor cells, or may otherwise decrease the size of a tumor, decrease metastases, or contribute to the lessening of any symptoms associated with a tumor.

[0025] It is not necessary that the vector comprise the complete nucleic acid sequence of the antigen or effector protein. It is only necessary that the antigen sequence used be capable of eliciting an immune response, or the effector protein be capable of eliciting the desired effect. The antigen or effector protein may be one that was not found in that exact form in the parent organism. For example, a sequence coding for an antigen or effector protein comprising 100 amino acid residues may be transferred in part into a recombinant bacterium so that a peptide comprising only 75, 65, 55, 45, 35, 25, 15, or even 10, amino acid residues is produced by the recombinant bacterium. Alternatively, if the amino acid sequence of a particular antigen, effector protein, or fragment thereof is known, it may be possible to chemically synthesize the nucleic acid fragment or analog thereof by means of automated nucleic acid sequence synthesizers, PCR, or the like and introduce said nucleic acid sequence into the appropriate copy number vector.

[0026] In another alternative, a vector may comprise a long sequence of nucleic acid encoding several nucleic acid sequence products, one or all of which may be antigenic or be effector proteins. In some embodiments, a vector described herein may comprise a nucleic acid sequence encoding at least one antigen or effector protein, at least two antigens or effector proteins, at least threeantigens or effector proteins, or more than three antigens or effector proteins. These antigens or effector proteins may be encoded by two or more open reading frames operably linked to be expressed coordinately as an operon, wherein each antigen or effector proteins is synthesized independently. Alternatively, the two or more antigens or effector proteins may be encoded by a single open reading frame such that the antigens or effector proteins are synthesized as a fusion protein.

[0027] In further embodiments, a nucleic acid sequence encoding an antigen or effector protein may comprise a secretion signal. In other embodiments, an antigen or effector protein may be toxic to the recombinant bacterium.

[0028] 2. Promoter Regulated by Repressor

[0029] The vector comprises a nucleic acid sequence encoding at least one antigen operably linked to a promoter regulated by the repressor, encoded by a chromosomally integrated nucleic acid sequence. One of skill in the art would recognize, therefore, that the selection of a repressor dictates, in part, the selection of the promoter operably linked to a nucleic acid sequence encoding an antigen or effector protein of interest. For instance, if the repressor is LacI, then the promoter may be selected from the group consisting of LacI responsive promoters, such as Ptrc, Plac, PT7lacand Ptac. If the repressor is C2, then the promoter may be selected from the group consisting of C2 responsive promoters, such as P22 promoters PL and PR. If the repressor is C1, then the promoter may be selected from the group consisting of C1 responsive promoters, such as λ promoters PLand PR.

[0030] In each embodiment herein, the promoter regulates expression of a nucleic acid sequence encoding the antigen or effector protein, such that expression of the nucleic acid sequence encoding an antigen or effector protein is repressed when the repressor is synthesized (i.e. during in vitro growth of the bacterium), but expression of the nucleic acid sequence encoding an antigen or effector protein is high when the repressor is not synthesized (i.e. in an animal or human host). The concentration of the repressor will decrease with every cell division after expression of the nucleic acid sequence encoding the repressor ceases. In some embodiments, the concentration of the repressor decreases enough to allow high level expression of the nucleic acid sequence encoding an antigen or effector protein after about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 divisions of the bacterium. In an exemplary embodiment, the concentration of the repressor decreases enoughto allow high level expression of the nucleic acid sequence encoding an antigen or expression protein after about 5 divisions of the bacterium in an animal or human host.

[0031] In certain embodiments, the promoter may comprise other regulatory elements. For instance, the promoter may comprise lacO if the repressor is LacI. This is the case with the lipoprotein promoter Plpp that is regulated by LacI since it possesses the LacI binding domain lacO.

[0032] In one embodiment, the repressor is a LacI repressor, and the promoter is Ptrc.

[0033] 3. Expression of the Nucleic Acid Sequence Encoding an Antigen or Effector Protein

[0034] As detailed above, the expression of the nucleic acid sequence encoding the antigen or effector protein should be repressed when the repressor is synthesized. For instance, if the repressor is synthesized during in vitro growth of the bacterium, expression of the nucleic acid sequence encoding the antigen or effector protein should be repressed. Expression may be “repressed” or “partially repressed” when it is about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or even less than 1% of the expression under non-repressed conditions. Thus, although the level of expression under conditions of “complete repression” might be exceeding low, it is likely to be detectable using very sensitive methods since repression can never by absolute. Conversely, the expression of the nucleic acid sequence encoding the antigen or effector protein should be high when the expression of the nucleic acid sequence encoding the repressor is repressed. For instance, if the nucleic acid sequence encoding the repressor is not expressed during growth of the recombinant bacterium in the host, the expression of the nucleic acid sequence encoding the antigen or effector protein should be high. As used herein, “high level” expression refers to expression that is strong enough to elicit an immune response to the antigen or to see the effects of the effector protein on the tumor cell. Consequently, the copy number correlating with high level expression can and will vary depending on the antigen or effector protein and the type of immune response desired.

[0035] Methods of determining whether an antigen elicits an immune response such as by measuring antibody levels or antigen-dependent T cell populations or antigen-dependent cytokine levels are known in the art, and methods of measuring levels of expression of antigen or effector protein encoding sequences by measuring levels of mRNA transcribed or by quantitating the level of antigen or effector protein synthesis are also known in the art.

[0036] B. Nucleic Acid Vaccine Vector

[0037] A recombinant bacterium provided herein may encompass a nucleic acid vaccine vector. Such a vector is typically designed to be transcribed in the nucleus of the host cell to produce mRNA encoding one or more antigens or effector proteins of interest. To increase performance, a nucleic acid vaccine vector should be targeted to the nucleus of a host cell and should be resistant to nuclease attack.

[0038] In one embodiment, a nucleic acid vaccine vector may be targeted to the nucleus using a DNA nuclear targeting sequence. It is well known that nonviral gene-delivery systems are promising tools for gene therapy and DNA vaccination applications. Compared with viral-based systems, these systems possess several advantages, including excellent safety profiles, an essentially unlimited DNA carrying capacity, and so forth. However, gene expression from such plasmids in vivo remains much lower, largely because of the inability of the DNA to effectively translocate through the nuclear pore complexes. One of the major mechanisms of nonviral nucleic acid vaccine vectors import into the nuclei in nondividing eukaryotic cells is dependent on DNA nuclear targeting sequences (DTS). The SV40 enhancer, which is known to bind to over 10 distinct transcription factors, is an excellent DTS. The minimum requirement for this function is a single copy of a 72-bp element of the SV40 enhancer, in combination with the CMV immediate- early gene enhancer / promoter (CMV E / P). To increase the efficiency of the nucleic acid vaccine vector delivery system, the SV 4072-bp repeat enhancer, was first inserted into the original nucleic acid vaccine vector to serve as a DTS (I).

[0039] Transcription factor NF-κB is found in almost all animal cell types. The binding affinity of NF-κB to their DNA-binding sites (κB sites) is high and the translocation of NF-κB-DNA complexes into the nucleus is rapid (minutes). Depending on their position relative to the encoding gene, the binding sites could also act as transcriptional enhancers that further increase gene- expression levels. Salmonella infection rapidly stimulates the synthesis of eukaryotic transcription factors, such as NF-κB and AP-2. It was postulated that the plasmid DNA with κB and AP-2 binding sites (DTS II) would allow newly synthesized NF-κB or AP-2, during Salmonella infection, to bind to the plasmid DNA in the cytoplasm and transport it to the nucleus through the protein nuclear import machinery.

[0040] In addition, nuclease degradation of nucleic acid vaccine vectors after delivery and during trafficking to the nucleus is another barrier that leads to inefficient DNA vaccination. A nucleic acid vaccine vector described herein may also be resistant to eukaryotic nuclease attack. Inparticular, the polyadenylation signal may be modified to increase resistance to nuclease attack. Suitable polyadenylation signals that are resistant to nuclease attack are known in the art. For instance, the SV40 late poly A signal may be used. Alternatively, other poly A adenylation signal sequences could be derived from other DNA viruses known to be successful in infecting avian and / or mammalian species.

[0041] II. Recombinant bacterium

[0042] A recombinant bacterium provided herein is generally an anaerobic bacterium. This bacterium may include one or more specific mutations aimed at enhancing its therapeutic efficacy. These mutations may serve to increase the bacterium's invasiveness, improve its localization within tumor tissues, and reduce its fitness in normal tissues.

[0043] Additionally, the bacterium may have mutations designed to boost host cell apoptosis induced by the bacterium, facilitate bacterial lysis, or attenuate the bacterium. The goal is to optimize its performance as a tumor therapy. Furthermore, the bacterium may contain vectors for the expression of nucleic acids, including those encoding antigens or effector proteins.

[0044] A. Retention of the Switch-On for Synthesis of Proteins Required for Invasiveness

[0045] A recombinant bacterium provided herein may also exhibit hyper-invasiveness. Here, “hyper-invasive” denotes a bacterium that can penetrate host and a tumor more effectively than a wild-type bacterium of the same strain. Invasion efficiency can be assessed using established techniques, such as measuring colony-forming units (CFUs) per gram of tumor tissue.

[0046] An early step in the establishment of S. Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine, mainly by bacteria invading M-cells overlying Peyer’s patches. The ability of Salmonella to invade intestinal cells of the host is also critical for efficient nucleic acid vaccine vector delivery. Most genes responsible for the Salmonella invasive phenotype are encoded on SPI-1, and their transcription is controlled by the hilA transcriptional activator. The expression of hilA is regulated by environmental signals, including oxygen, osmolarity, pH, and growth phase, such that the presence of any one suboptimal condition results in repression of hilA expression and the invasive phenotype. On the other hand, although it was thought that Salmonella resides and proliferates within a membrane-bound vacuole in epithelial cells, it has recently been discovered that there are at least two transcriptionally distinct intracellular populations of replicating bacteria in epithelial cells: T3SS-2–induced intravacuolar bacteria and T3SS-1–induced flagellated bacteria that are invasion-primed, cytosolic, fast-replicating, moving freely within the cells, and well prepared to spread and invade new cells. Increase of the subpopulation of invasion-primed cytosolic Salmonella would greatly benefit nucleic acid vaccine vector delivery.

[0047] A recombinant bacterium may comprise a mutation that increases expression of hilA. For instance, the promoter of hilA may be mutated to enable constitutive expression of hilA, such as ΔPhilA::PtrcΔlacOhilA mutation. Such a mutation replaces the wild-type hilA promoter with the Ptrcpromoter that lacks the lacO operator sequence. This allows constitutive expression of hilA, even when repressor LacI is expressed.

[0048] B. Engineering GMSs to Reduce Salmonella Induced Host Cell Pyroptosis / Apoptosis

[0049] Salmonella strains induce host cell death during infection by several mechanisms and this is likely to diminish transcription of a nucleic acid vaccine vector after trafficking to the nucleus. Salmonella grown under conditions to express the SPI-1 T3SS activate the NLRC4 inflammasome in macrophages, which activates caspase-1 leading to cell death by a process termed pyroptosis. In contrast, Salmonella-induced epithelial cell death, which has features of delayed classic apoptosis, depends on both the SPI-2 T3SS and the spv locus even though Salmonella invasion involves the SPI-1 T3SS.

[0050] One of the major SPI-2 T3SS-secreted effectors that induce apoptosis have been identified: SseL. SseL, a Salmonella deubiquitinase, has been shown to be involved in macrophage cytotoxicity. In addition, SseL deubiquitinates IκBα, the major regulator of the classic NF-κB activation pathway. NF-κB activation is significantly increased after infection of macrophages with an SseL mutant, and this activity can be decreased to wild-type levels by complementation with a low copy plasmid synthesizing SseL. These results show that the action of SseL is to decrease NF-κB signaling, thereby decreasing the antiapoptotic and proinflammatory effects of this pathway and support the idea that SseL may also act in concert with other effectors to decrease activation of innate immunity. Deletion of sseL from GMS strains would reduce Salmonella- induced apoptosis, enhance innate immune responses by enhancing NF-κB activation, and simultaneously enhance NF-κB-mediated nuclear targeting of the DNA vector.

[0051] In addition, an interesting putative virulence regulator is the thermo-sensing gene regulator TlpA. TlpA can alter its DNA-binding according to variation in temperature, and concomitantly, its DNA regulatory characteristics functions. Strong homologies to the operator sequence oftlpAare found in front of defined virulence genes, such as the spvABCD genes. Like the spv genes,tlpAis located on the large virulence-associated plasmid and is conserved in Salmonella carrying the virulence plasmid. Thus, the question is whether deletion oftlpAcan reduce Salmonella-induced apoptosis to enhance nucleic acid vaccine vector delivery. Elimination of synthesis of TlpA and SseL reduce apoptosis in mice infected with Salmonella.

[0052] C. Lysis

[0053] A recombinant bacterium disclosed herein is capable of regulated lysis. Lysis of the bacterium within the host cell may release a bolus of antigen, or alternatively, may release a nucleic acid vaccine vector for transcription by the tumor cell. Lysis also provides a means of biocontainment. For instance, the bacterium may comprise a mutation in a nucleic acid sequence encoding a protein involved in muramic acid synthesis, such as murA. It is not possible to alter murA by deletion, however, because a ΔmurA mutation is lethal and cannot be isolated. This is because the missing nutrient required for viability is a phosphorylated muramic acid that cannot be exogenously supplied since enteric bacteria cannot internalize it. Consequently, the murA nucleic acid sequence may be altered to make expression of murA dependent on a nutrient (e.g., arabinose) that can be supplied during the growth of the bacterium. For example, the alteration may comprise a ΔPmurA::TT araC PBADmurA deletion-insertion mutation. During in vitro growth of the bacterium, this type of mutation makes synthesis of muramic acid dependent on the presence of arabinose in the growth medium. During growth of the bacterium in a host, however, arabinose is absent. Consequently, the bacterium is non-viable and / or avirulent in a host unless the bacterium further comprises at least one extrachromosomal vector comprising a nucleic acid sequence, that when expressed, substantially functions as murA. Recombinant bacteria with a ΔPmurA::TT araC PBADmurA deletion-insertion mutation grown in the presence of arabinose exhibit effective colonization of effector lymphoid tissues after oral administration prior to cell death due to cell wall-less lysing.

[0054] Similarly, a recombinant bacterium may comprise the araC PBAD c2 cassette inserted into the asdA nucleic acid sequence that encodes aspartate semialdehyde dehydrogenase, a necessary enzyme for DAP synthesis, a required component of the peptidoglycan layer of the bacterial cell wall. The chromosomal asdA nucleic acid sequence is typically inactivated to enable use of plasmid vectors encoding the wild-type asdA nucleic acid sequence in the balanced-lethal host- vector system. This allows stable maintenance of plasmids in vivo in the absence of any drug resistance attributes that are not permissible in live bacterial vaccines.

[0055] The bacterium may comprise a mutation in the murA nucleic acid sequence encoding the first enzyme in muramic acid synthesis and the asdA nucleic acid sequence essential for DAP synthesis. By way of non-limiting example, these embodiments may comprise the chromosomal deletion-insertion mutations ΔasdA::TT araC PBAD c2 and ΔPmurA::TT araC PBAD murA. This host- vector grows in LB broth with 0.1% L-arabinose but is unable to grow in or on media devoid of arabinose since it undergoes cell wall-less death by lysis. The onset of programmed lysis may be delayed about one cell division by including a Δ(araC PBAD)::P22 PRaraBAD mutation, which initially prevents breakdown of accumulated arabinose at the time of inoculation.

[0056] Bacterium that comprises these mutations also comprise a plasmid that contains a nucleic acid sequence that substitutes for murA and asdA. This allows the bacterium to grow in permissive environments, e.g. when arabinose is present. For instance, vectors pYA3681 and pYA4545 contain the murA nucleic acid sequence (with altered start codon sequences from ATG to GTG to decrease translation efficiency) under the control of an araC PBADpromoter. The second nucleic acid sequence under the direction of this promoter is the asdA nucleic acid sequence (with altered start codon sequences from ATG to GTG to decrease translation efficiency). The P22 PR promoter is in the anti-sense direction of both the asdA nucleic acid sequence and the murA nucleic acid sequence. The P22 PRis repressed by the C2 repressor made during growth of the strain in media with arabinose (due to the ΔasdA::TT araC PBAD c2 deletion-insertion). However, C2 concentration decreases due to cell division in vivo to cause PRdirected synthesis of anti-sense mRNA to further block translation of asdA and murA mRNA. The araC PBADsequence is also not from E. coli B / r as originally described but represents a sequence derived from E. coli K-12 strain χ289 with tighter control and less leakiness in the absence of arabinose. In the preferred embodiment, transcription terminators (TT) flank all the domains for controlled lysis, replication, and expression so that expression in one domain does not affect the activities of another domain. As a safety feature, the plasmid asdA nucleic acid sequence does not replace the chromosomal asdA mutation since they have a deleted sequence in common. Additionally, the E. coli murA nucleic acid sequence was used in the plasmid instead of using the Salmonella murA nucleic acid sequence. In addition to being fully attenuated, this construction exhibits complete biological containment. This property enhances safety and minimizes the potential for exposure of individuals not intended for tumor treatment.

[0057] In addition, the ΔaraBAD denotes the deletion of structural genes for catabolism of arabinose, thereby preventing the use of arabinose retained in the cell cytoplasm at the time of immunization. The ΔaraE mutation, which deletes the gene for arabinose transport, enhances retention of arabinose by precluding its leakage from the cell. This inability to use arabinose prolongs time to lysis in vivo by one to two cell divisions, allowing increasing cell numbers and thus enhancing antigen delivery. The ΔaraBAD and ΔaraE mutations were included in the original lysis strains to create GMS strains, which exhibit delayed lysis phenotype.

[0058] One of skill in the art will recognize that other nutrients besides arabinose may be used in the above mutations. By way of non-limiting example, xylose, mannose, and rhamnose regulatory systems may also be used. Additionally, a bacterium may comprise a mutation in a protein involved in GDP-fucose synthesis to preclude formation of colonic acid. Nonlimiting examples of such a mutation include Δ(gmd-fcl). A bacterium may also comprise a mutation like ΔrelA that uncouples cell wall-less death from dependence on protein synthesis.

[0059] Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. Δ(wza-wcaM), which deleted the whole operon for CA synthesis, developed higher vaginal IgA titers against the heterologous protective antigen and higher levels of antigen- specific IgA secretion cells in lungs. This strain also resulted in better gamma interferon (IFN-γ) responses than the strain without this deletion.

[0060] A recombinant bacterium may also comprise a ΔrelA::TT araC PBADlacI TT deletion- insertion mutation so that growth of the strain in the presence of arabinose causes synthesis of LacI to initially repress synthesis of protein antigens encoded by sequences under the control of Ptrc. Because of cell division in vivo during colonization of lymphoid tissues, LacI becomes diluted and expression of Ptrc controlled genes commences with synthesis of the protective antigen to stimulate induction of immune responses. In all cases the regulated delayed lysis phenotype is totally attenuating with no persistence of bacteria cells in vivo and no survival of bacteria cells if excreted.

[0061] Lysis of the bacterium will typically release lipid A, an endotoxin. A bacterium may comprise a mutation that reduces the toxicity of lipid A. A lysis of the bacterium may include a mutation that causes synthesis of the mono-phosphoryl lipid A. This form of lipid A is non-toxic, but still serves as an adjuvant agonist. For instance, a recombinant bacterium may comprise a ΔpagP::Plpp lpxE mutation.

[0062] Moreover, a bacterium comprising a nucleic acid vaccine vector may also comprise a mutation that eliminates the periplasmic endonuclease I enzyme, such as a ΔendA mutation. This type of mutation is designed to increase vector survival upon the vector's release into the host cell.

[0063] D. Enable S. Typhimurium to escape the endosome after invasion

[0064] Salmonella strains induce intestinal epithelial cells to take them up into a Salmonella- containing vacuole (SCV) and then manipulate the intracellular trafficking of the vacuole to promote survival and replication of the pathogen. The sifA gene is a Salmonella pathogenicity island 2 (SPI-2)- encoded, type III secretion system (T3SS)-secreted effector protein that governs conversion of the SCV into filaments. Deletion of sifA releases Salmonella into the cytosol. An GMS-regulated delayed lysis strain harboring the sifA deletion mutation escaped from the endosome before GMS lysis, allowing class I presentation of antigen or nucleic acid vaccine vector delivery.

[0065] E. Maximized Localization in Tumors

[0066] A recombinant bacterium may comprise a mutation to increase the expression of a nucleic acid encoding a chemoreceptor that directs chemotaxis towards tumors or increases penetration of tumors. For instance, the expression of the nucleic acid encoding the aspartate and maltose receptor, e.g. tar, may be increased. In particular, the promoter of the nucleic acid encoding the receptor may be replaced with a constitutive promoter. A bacterium may comprise a ΔPtar::PtrcΔlacOtar mutation. This allows constitutive expression of Tar, even when repressor LacI is expressed. The expression of the nucleic acid encoding the serine receptor, e.g. tsr, may be increased. In particular, the promoter of the nucleic acid encoding the receptor may be replaced with a constitutive promoter. A bacterium may comprise a ΔPtsr::Ptrc ΔlacOtsr mutation. This allows constitutive expression of Tsr, even when repressor LacI is expressed.

[0067] A recombinant bacterium may comprise a mutation that decreases the expression of a nucleic acid encoding a chemoreceptor that directs chemotaxis towards necrosis. This allows bacterial accumulation in a quiescent tumor, as opposed to necrotic cells. For instance, the expression of the nucleic acid encoding the ribose / galactose receptor, e.g. trg, may be decreased. In particular, the trg sequence may be deleted or mutated to prevent or decrease expression of the nucleic acid or translation of the nucleic acid into the corresponding protein. Suitable mutations may include the Δtrg mutation, which will result in cessation of Trg synthesis in vivo due to the lack of rhamnose.

[0068] A bacterium may comprise both a mutation that increases the expression of one or more nucleic acids that encode a chemoreceptor and a mutation that decreases the expression of one or more different nucleic acids that encode a chemoreceptor.

[0069] III. Bacterial host-vector systems

[0070] In an exemplary embodiment, a bacterium may comprise one or more mutations to increase invasiveness, one or more mutations that enhance stimulation of host innate immune responses, one or more mutations to increase bacterium-induced host programmed cell death, one or more mutations to induce lysis of the bacterium, one or more vectors to express a nucleic acid encoding an antigen or effector protein, one or more mutations to attenuate the bacterium, and one or more mutations to enhance the performance of the bacterium as a vaccine, one or more mutations to enhance the localization in tumors.

[0071] A. Self-destructing bacterial host-vector systems harboring lysis vector pYA3681 for host antigens, effectors, and therapeutics delivery

[0072] In one embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(gmd-fcl) ΔrelA ΔendA.

[0073] In another embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 ΔaraBAD Δpmi ΔrelA::araC PBADlacI Δgmd-fcl ΔrelA::araC PBADlacI.

[0074] In still another embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(araC PBAD)::P22 PRaraBAD Δ(wza-wcaM) ΔrelA::araC PBADlacI TT ΔpagP::PlpplpxE ΔendA.

[0075] In a further embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(araC PBAD)::P22 PR araBAD Δ(wza-wcaM) Δpmi ΔrelA::araC PBAD lacI TT ΔpagP::Plpp lpxE ΔendA.

[0076] B. Self-destructing and tumor-navigating bacterial host-vector systems harboring lysis vector pYA3681 for TME-targeted antigens, effectors, and therapeutics delivery

[0077] In one embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(araC PBAD)::P22 PRaraBAD Δ(wza-wcaM) Δpmi ΔrelA::araC PBADlacI TT ΔpagP::PlpplpxE ΔendA Δ Ptar::PtrcΔlacO tar.

[0078] In another embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(araC PBAD)::P22 PRaraBAD Δ(wza-wcaM) Δpmi ΔrelA ΔpagP::PlpplpxE ΔendA ΔPtar::Ptrc ΔlacOtar Δtrg.

[0079] In still another embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(araC PBAD)::P22 PRaraBAD Δ(wza-wcaM) Δpmi ΔrelA::araC PBADlacI TT ΔpagP::PlpplpxE ΔendA ΔPtar::Ptrc ΔlacOtar ΔPtsr::Ptrc ΔlacOtsr.

[0080] In yet another embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(araC PBAD)::P22 PRaraBAD Δ(wza-wcaM) Δpmi ΔrelA::araC PBADlacI TT ΔpagP::PlpplpxE ΔendA ΔPtar::PtrcΔlacOtar ΔPtsr::Ptrc ΔlacO tsr and Δtrg.

[0081] In an further embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(wza-wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::Ptrc ΔlacO hilA ΔPtar::Ptrc ΔlacO tar, and Δtrg.

[0082] In still further embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(wza-wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::Ptrc ΔlacOhilA ΔPtar::Ptrc ΔlacOtar, ΔPtsr::Ptrc ΔlacOtsr and Δtrg.

[0083] In yet further embodiment, a lysis vector pYA3681-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(wza-wcaM) Δpmi ΔrelA ΔpagP::Plpp lpxE ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::PtrcΔlacOhilA ΔPtar::PtrcΔlacOtar, ΔPtsr::PtrcΔlacOtsr and Δtrg.

[0084] C. Self-destructing bacterial host-vector systems harbor nucleic acid vaccine vector pYA4545 encoding antigens, effectors, and therapeutics for TME-targeted delivery

[0085] In one embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 ΔaraBAD ΔaraE Δ(gmd-fcl) ΔrelA ΔsifA ΔendA ΔPhilA::Ptrc ΔlacO hilA, ΔtlpA, and ΔsseL.

[0086] In another embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(wza- wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔPhilA::Ptrc ΔlacOhilA ΔtlpA, and ΔsseL.

[0087] In still another embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(wza-wcaM) Δpmi ΔrelA ΔpagP::PlpplpxE ΔsifA ΔendA ΔPhilA::Ptrc ΔlacOhilA ΔtlpA, and ΔsseL.

[0088] D. Self-destructing and tumor-navigating bacterial host-vector systems harbor nucleic acid vaccine vector pYA4545 encoding antigens, effectors, and therapeutics for TME-targeted delivery

[0089] In one embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(wza- wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔPhilA::Ptrc ΔlacOhilA ΔsseL ΔtlpAΔPtar::Ptrc ΔlacOtar.

[0090] In another embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(wza- wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔPhilA::PtrcΔlacOhilA ΔsseL ΔtlpAΔPtar::PtrcΔlacOtar, and ΔPtsr::PtrcΔlacOtsr.

[0091] In still another embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(wza-wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔPhilA::Ptrc ΔlacOhilA ΔsseL ΔtlpAΔPtar::Ptrc ΔlacOtar, and Δtrg.

[0092] In yet another embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(wza-wcaM) Δpmi ΔrelA ΔsifA ΔendA ΔPhilA::Ptrc ΔlacOhilA ΔsseL ΔtlpAΔPtar::Ptrc ΔlacOtar, ΔPtsr::PtrcΔlacOtsr, and Δtrg.

[0093] In a further embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBADmurA ΔasdA::TT araC PBADc2 Δ(wza- wcaM) Δpmi ΔrelA ΔpagP::Plpp lpxE ΔsifA ΔendA ΔPhilA::PtrcΔlacOhilA ΔsseL ΔtlpAΔPtar::PtrcΔlacOtar, ΔPtsr::PtrcΔlacOtsr, and Δtrg.

[0094] In still a further embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(wza-wcaM) Δpmi ΔrelA ΔrecF ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::PtrcΔlacOhilA ΔPtar::Ptrc ΔlacO tar.

[0095] In yet a further embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2Δ(wza-wcaM) Δpmi ΔrelA ΔrecF ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::Ptrc ΔlacO hilA ΔPtar::Ptrc ΔlacO tar, and ΔPtsr::PtrcΔlacOtsr.

[0096] In an alternative embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBAD c2 Δ(wza-wcaM) Δpmi ΔrelA ΔrecF ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::PtrcΔlacOhilA ΔPtar::Ptrc ΔlacO tar, ΔPtsr::Ptrc ΔlacOtsr, and Δtrg.

[0097] In still an alternative embodiment, a nucleic acid vaccine vector pYA4545-harboring bacterium may comprise the following mutations: ΔPmurA::TT araC PBAD murA ΔasdA::TT araC PBADc2 Δ(wza-wcaM) Δpmi ΔrelA ΔpagP::PlpplpxE ΔrecF ΔsifA ΔendA ΔsseL ΔtlpAΔPhilA::PtrcΔlacOhilA ΔPtar::Ptrc ΔlacOtar, ΔPtsr::Ptrc ΔlacOtsr, and Δtrg.

[0098] Some embodiments of the instant disclosure comprise a species or subspecies of the Salmonella genera. For instance, the recombinant bacterium may be a Salmonella Enterica serovar. A bacterium of the disclosure may be derived from (i.e., an isolate of) S. Enterica serovar Typhimurium, referred to herein as Salmonella Typhimurium, or from Salmonella Typhi, Salmonella Paratyphi, Salmonella Enteritidis, Salmonella Choleraesius, Salmonella Arizona, or Salmonella Dublin. As used herein, “S. Typhimurium” refers to an isolate of S. Typhimurium. Likewise, the terms “S. Typhi,” “S. Paratyphi,” “S. Enteritidis,” “S. Choleraesius,” “S. Arizona,” and “S. Dublin” as used herein refer to isolates of S. Typhi, S. Paratyphi, S. Enteritidis, S. Choleraesius, S. Arizona, and S. Dublin, respectively. As used herein the terms “strain” and “isolate” are used interchangeably. In preferred embodiments, the GMS bacterium may be S. Typhimurium.

[0099] As used herein, the terms “genetically modified” and “genetically engineered” are used interchangeably and refer to a prokaryotic cell that includes an exogenous polynucleotide, regardless of the method used for insertion. In some cases, the cell has been modified to comprise a non-naturally occurring nucleic acid molecule that has been created or modified by the hand of man (e.g., using recombinant DNA technology) or is derived from such a molecule (e.g., by transcription, translation, etc.). A cell that contains an exogenous, recombinant, synthetic, and / or otherwise modified polynucleotide is considered to be an engineered cell. The term “altered,” as used herein, refers to any change in the nucleic acid sequence that results in the nucleic acid sequence not being expressed. The alteration may result in the nucleic acid sequence not being expressed in a host. The alteration may be a deletion. Alternatively, the alteration may place anessential nucleic acid under the control of a regulatable promoter, such that the nucleic acid is not expressed in a host.

[0100] The “∆” as used herein, refers to gene deletion; The “::” as used herein, refers to gene insertion; The “asdA” as used herein, refers to a gene encoding aspartate-semialdehyde dehydrogenase. The asdA mutants (“ΔasdA”) of Gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. The “murA” refers to a gene required for the synthesis of the peptidoglycan layer of the bacterial cell wall. Like asdA mutants, murA mutants (“ΔmurA”) are deficient in bacterial cell wall synthesis.

[0101] The GMS bacterium may further comprise a therapeutic or a vaccine.

[0102] In another aspect, provided herein is a method for treating a subject in need thereof, the method comprising administering a genetically modified Salmonella bacterium comprising a therapeutic or vaccine described herein. The bacterium may be administered orally or via intra- tumoral injection. The bacterium may be administered orally or via intra-tumoral injection. The term “bacterium” as used herein refers to a single bacterium or “bacteria”, which includes a culture or population of the bacterium.

[0103] In certain embodiments, a genetically modified bacterium of the disclosure may also be attenuated. As used herein, the term “attenuated” refers to the state of the bacterium wherein the bacterium has been weakened from its wild-type fitness by some form of recombinant or physical manipulation such that the bacterium’s virulence is reduced relative to a control (a non- recombinant / non-manipulated bacterium). This includes altering the genotype of the bacterium to reduce its ability to cause disease. However, the bacterium’s ability to colonize the tumor is, preferably, not substantially compromised. For instance, regulated attenuation allows the recombinant bacterium to express one or more nucleic acids encoding products important for the bacterium to withstand stresses encountered in the host after immunization. This allows efficient invasion and colonization of tumor tissues before the genetically modified bacterium is regulated to display the attenuated phenotype. As used herein in this context, the term “reduce / reduced” means a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, still more preferably 80%, even more preferably 90% and most preferably of 100% as compared to the appropriate control.

[0104] The genetically modified Salmonella described herein can be used in a variety of applications. For example, the genetically modified Salmonella can be used in therapeutic methods to treat cancer or a cancer-associated condition. In some cases, a method of treating cancer in a subject in need thereof will comprise administering an effective amount of a modified Salmonella bacterium having the genetic modifications described herein and, thus, being tumor navigating, self-eradicating, and host immune system evading, whereby the genetically modified Salmonella bacterium treats cancer in the subject.

[0105] As used herein, the term “effective amount” means, in the context of a composition, an amount of an immunogenic composition capable of inducing an immune response that reduces the incidence of or lessens the severity of infection or incident of disease in an animal. Alternatively, in the context of a therapy, the term “effective amount” refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity or duration of a disease or disorder (e.g., cancer), or one or more symptoms thereof, prevent the advancement of a disease or disorder, cause the regression of a disease or disorder, prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disease or disorder, or enhance or improve the prophylaxis or treatment of another therapy or therapeutic agent. The effective amount to be administered will depend upon the host receiving the modified bacteria as well as factors such as the size, weight, and age of the host.

[0106] As used herein, “subject” or “host” refers to an animal or a patient for whom the described treatment is intended. In exemplary embodiments, subjects treated according to the methods provided herein are human. In other cases, subjects treated according to the methods provided herein are non-human mammals, including by way of example and not limitation, members of rodentia (e.g., mouse, rat, guinea pig), lagomorpha (e.g., rabbits, hares), perissodactyla (e.g., horses, donkeys, etc.), artodactyla (e.g., pigs, cows, sheep), carnivora (e.g., cats, canines), and primates (e.g., apes, monkeys, baboons, and humans).

[0107] As used herein, the terms "treat" and "treating" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to treat, rescue, ameliorate, or otherwise lessen an undesired symptom or condition associated with cancer or any condition associated with aberrant cell proliferation. In some cases, the term "treated" refers to any beneficial effect on the progression of a disease or condition. Beneficial effects can include reversing, alleviating, inhibiting the progress of, preventing, or reducing the likelihood of the disease orcondition to which the term applies or one or more symptoms or manifestations of such a disease or condition. Where the disease or condition is cancer or a cancer-associated condition, treating can refer to the management and care of a patient for the purpose of combating cancer, and can include reversing, alleviating, inhibiting the progress of, preventing, or reducing the likelihood of, or lessening the severity of any aspect of the cancer or cancer-associated condition (e.g., metastasis, tumor growth). As used herein, the terms "preventing" and "prevent" refer not only to a complete prevention of a certain disease or condition, but also to partially or substantially attenuating, reducing the risk of, or delaying the development or recurrence of the disease or condition to which the term applies.

[0108] In some cases, the methods provided herein are directed to treating or preventing a cancer in a subject by administering a composition provided herein. In other cases, the present disclosure provides a method of inhibiting, retarding, or preventing the growth of a tumor or tumor cells in a subject. Examples of cancers appropriate for methods of treating or preventing as provided herein include, without limitation, lung cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, kidney cancer, ovarian cancer, colon cancer, colorectal cancer, breast cancer, cervical cancer, brain cancer, esophageal cancer, and stomach cancer. Other diseases or conditions appropriate for methods of treating or preventing as provided herein include, without limitation, lymphoma and chronic and acute leukemia.

[0109] Any appropriate route or mode of administration to the subject can be employed according to a method provided herein. In some cases, administering comprises oral administration of the genetically modified Salmonella bacterium. In other cases, administering comprises intra- tumoral injection of the genetically modified Salmonella bacterium. The mode of administration can be determined based on the physical location, type, or the number of tumors in the subject's body.

[0110] Clinicians, physicians, and other health care professionals can administer genetically modified Salmonella bacteria to a subject in need thereof according to a method provided herein. In some cases, a single administration of the composition may be sufficient. In other cases, more than one administration of the composition is performed at various intervals (e.g., once per week, twice per week, daily, monthly) or according to any other appropriate treatment regimen. The duration of treatment can be a single dose or periodic multiple doses for as long as the administration of a composition provided herein is tolerated by the subject.

[0111] Any appropriate method can be practiced to determine, detect, or monitor a subject's response to treatment according to a method provided herein. As used herein, "determining a subject's response to treatment" refers to the assessment of the results of a therapy in a subject in response to administration of a composition provided herein or to treatment according to a method provided herein. For example, a subject's condition can be monitored continuously or evaluated at appropriate time intervals (e.g., at regular or irregular time points) to detect and / or monitor any changes in disease progression (e.g., change in tumor size) as an indicator of the subject's response to a composition comprising genetically modified Salmonella bacteria as described herein. In some cases, tumors can be measured to detect or monitor any change in, for example, tumor size or tumor growth rate (e.g., tumor expansion or shrinkage, inhibited or accelerated tumor growth rate). For example, detection methods such as computed tomography (CT), magnetic resonance imaging (MRI) scanning, and x-ray (e.g., chest x-ray) can be used. In some cases, ultrasound examinations can be used to detect and measure tumor regression or to detect the progression of lesions. In other cases, evaluation of a tumor can involve cytology or histology of, for example, biopsy samples. For solid tumors, evaluation of a subject's response to treatment as provided herein can include assessing RECIST ("Response Evaluation Criteria in Solid Tumors"). RECIST criteria can be used to evaluate a subject's response to the therapy used to treat their disease or condition. See, for review, Therasse et al., J. Natl. Cancer Inst.92:205-16, 2000.

[0112] The term “promoter”, as used herein, may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of the same.

[0113] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), etc. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecules. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or include non- naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- aminoadeno sine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)- methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′- deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

[0114] Nucleic acids and / or other constructs of the disclosure may be isolated. As used herein, “isolated” means to separate from at least some of the components with which it is usuallyassociated whether it is derived from a naturally occurring source or made synthetically, in whole or in part.

[0115] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.

[0116] Nucleic acids, proteins, and / or other moieties of the disclosure may be purified. As used herein, purified means separate from the majority of other compounds or entities. A compound or moiety may be partially purified or substantially purified. Purity may be denoted by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.

[0117] In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. It is understood that certain adaptations of the disclosure described in this disclosure are a matter of routine optimization for those skilled in the art, and can be implemented without departing from the spirit of the disclosure, or the scope of the appended claims.

[0118] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0119] The terms “comprising”, “comprises” and “comprised of as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open- ended and do not exclude additional, non-recited members, elements, or method steps. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof, is meant to encompass the items listed thereafter and additional items. Embodiments referenced as “comprising” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements. Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed. Ordinal terms are used merely as labels to distinguish one claim element having a certain name from another element having the same name (but for the use of the ordinal term), to distinguish the claim elements.

[0120] The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 10%, and preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0121] The term “identity”, as recognized by those skilled in the art, represents a comparison between two or more amino acid sequences performed using published methods and software known in the art. For example, the compared amino acid sequences are optimally aligned, and the number of amino acid differences are counted and converted to a percentage. For example, if a first amino acid sequence of 50 amino acids is optimally aligned with a second amino acid sequence of 50 amino acids, and 5 out of 50 amino acids differ from the second amino acid sequence, then the first amino acid sequence is said to have 10% identity with the second amino acid sequence.

[0122] The mode of administration of a composition or combination, useful in a method of treatment provided herein, to an individual (such as a human) in need of thereof may be anymode known in the art to be suitable for delivering a pharmaceutical composition. A mode of administration may include but is not limited to, intravenously, intraperitoneally, subcutaneously, intramuscularly, by perfusion, and by peristaltic techniques. A composition or combination, useful in a method of treatment provided herein, may also be combined with other treatments known to those skilled in the art. For example an additional treatment for a cancer may be provided, including but not limited to chemotherapeutic treatment and radiation therapies.

[0123] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0124] The present disclosure has described one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the disclosure.Table 1. Informal Sequence Listing

[0125] Various exemplary embodiments of compositions and methods according to this disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims. 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 disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

[0126] EMBODIMENTS

[0127] Embodiment 1. A genetically modified Salmonella bacterium comprising a ΔPmurA::TT araC PBADmurA mutation; and a ΔasdA::TT araC PBADc2 mutation.

[0128] Embodiment 2. The bacterium of embodiment 1, further comprising mutations: Δ(gmd-fcl); ΔrelA; and ΔendA.

[0129] Embodiment 3. The bacterium of embodiment 1, further comprising mutations: ΔaraBAD; Δpmi; ΔrelA::araC PBADlacI; and Δgmd-fcl ΔrelA::araC PBADlacI.

[0130] Embodiment 4. The bacterium of embodiment 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM) ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp IpxE; and ΔendA.

[0131] Embodiment 5. The bacterium of embodiment 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; and ΔendA.

[0132] Embodiment 6. The bacterium of embodiment 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; ΔendA; and Δ Ptar::PtrcΔlacOtar.

[0133] Embodiment 7. The bacterium of embodiment 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔendA; ΔPtar::PtrcΔlacOtar; and Δtrg.

[0134] Embodiment 8. The bacterium of embodiment 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; ΔendA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr.

[0135] Embodiment 9. The bacterium of embodiment 1, further comprising mutations: Δ(araC PBAD)::P22 PRaraBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBADlacI TT; ΔpagP::PlpplpxE; ΔendA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

[0136] Embodiment 10. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; and Δtrg.

[0137] Embodiment 11. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::Ptrc ΔlacO tar; ΔPtsr::Ptrc ΔlacOtsr; and Δtrg.

[0138] Embodiment 12. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::Ptrc ΔlacOtar; ΔPtsr::Ptrc ΔlacOtsr; and Δtrg.

[0139] Embodiment 13. The bacterium of any one of embodiments 1-12, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 1.

[0140] Embodiment 14. The bacterium of embodiment 1, further comprising mutations: ΔaraBAD; ΔaraE; Δ(gmd-fcl); ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL.

[0141] Embodiment 15. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL.

[0142] Embodiment 16. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL.

[0143] Embodiment 17. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar.

[0144] Embodiment 18. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr.

[0145] Embodiment 19. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; and Δtrg.

[0146] Embodiment 20. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

[0147] Embodiment 21. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

[0148] Embodiment 22. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar.

[0149] Embodiment 23. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr.

[0150] Embodiment 24. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacO hilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacO tsr; and Δtrg.

[0151] Embodiment 25. The bacterium of embodiment 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

[0152] Embodiment 26. The bacterium of any one of embodiments 14-25, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 2.

[0153] Embodiment 27. The bacterium of any one of embodiments 1-26, further comprising a recombinant polynucleotide encoding at least one of an antigen and an effector protein.

[0154] Embodiment 28. A method of treating a subject in need thereof, the method comprising administering the bacterium of embodiment 27.

[0155] Embodiment 29. The bacterium of any one of embodiments 1-12 and 14-25, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 1 or 2.

Claims

CLAIMS We claim:

1. A genetically modified Salmonella bacterium comprising a ΔPmurA::TT araC PBADmurA mutation; and a ΔasdA::TT araC PBAD c2 mutation.

2. The bacterium of claim 1, further comprising mutations: Δ(gmd-fcl); ΔrelA; and ΔendA.

3. The bacterium of claim 1, further comprising mutations: ΔaraBAD; Δpmi; ΔrelA::araC PBAD lacI; and Δgmd-fcl ΔrelA::araC PBAD lacI.

4. The bacterium of claim 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM) ΔrelA::araC PBADlacI TT; ΔpagP::PlppIpxE; and ΔendA.

5. The bacterium of claim 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; and ΔendA.

6. The bacterium of claim 1, further comprising mutations: Δ(araC PBAD)::P22 PRaraBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBADlacI TT; ΔpagP::PlpplpxE; ΔendA; and Δ Ptar::PtrcΔlacOtar.

7. The bacterium of claim 1, further comprising mutations: Δ(araC PBAD)::P22 PRaraBAD; Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::PlpplpxE; ΔendA; ΔPtar::PtrcΔlacOtar; and Δtrg.

8. The bacterium of claim 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBADlacI TT; ΔpagP::PlpplpxE; ΔendA; ΔPtar::PtrcΔlacOtar; ΔPtsr::Ptrc ΔlacOtsr.

9. The bacterium of claim 1, further comprising mutations: Δ(araC PBAD)::P22 PR araBAD; Δ(wza-wcaM); Δpmi; ΔrelA::araC PBAD lacI TT; ΔpagP::Plpp lpxE; ΔendA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

10. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; and Δtrg.

11. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::Ptrc ΔlacOtar; ΔPtsr::Ptrc ΔlacOtsr; and Δtrg.

12. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::PlpplpxE; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::Ptrc ΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

13. The bacterium of any one of claims 1-12, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO:

1.

14. The bacterium of claim 1, further comprising mutations: ΔaraBAD; ΔaraE; Δ(gmd-fcl); ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL.

15. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL.

16. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔtlpA; and ΔsseL.

17. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar.

18. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr.

19. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; and Δtrg.

20. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

21. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔsifA; ΔendA; ΔPhilA::PtrcΔlacOhilA; ΔsseL; ΔtlpA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

22. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar.

23. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr.

24. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacO hilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacO tsr; and Δtrg.

25. The bacterium of claim 1, further comprising mutations: Δ(wza-wcaM); Δpmi; ΔrelA; ΔpagP::Plpp lpxE; ΔrecF; ΔsifA; ΔendA; ΔsseL; ΔtlpA; ΔPhilA::PtrcΔlacOhilA; ΔPtar::PtrcΔlacOtar; ΔPtsr::PtrcΔlacOtsr; and Δtrg.

26. The bacterium of any one of claims 14-25, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 2.

27. The bacterium of any one of claims 1-26, further comprising a recombinant polynucleotide encoding at least one of an antigen and an effector protein.

28. A method of treating a subject in need thereof, the method comprising administering the bacterium of claim 27.

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