Genetically modified salmonella strains for evading host immune response

Abstract

The present disclosure provides a Salmonella therapeutic delivery system having a modified surface for evading the host immune system, and methods of use thereof.

Description

GENETICALLY MODIFIED SALMONELLA STRAINS FOR EVADING HOSTIMMUNE RESPONSECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 708,961 filed on October 18, 2024, the content of which is incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (11262401535. xml; Size: 51,025 bytes; and Date of Creation: September 22, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Genetically modified Salmonella (GMS) strains that direct synthesis of protective antigens have been developed to provide novel, needle-free, low-cost protection against various diseases. GMS strains are constructed so that, following oral immunization, attenuation does not diminish their abilities to survive stresses encountered in the GI tract nor to colonize lymphoid tissues. Attenuation precludes GMS strains from causing disease, but they are fully capable of eliciting immune responses to result in long-lasting protection.

[0004] The inventors have previously developed GMS strains that feature regulated delayed lysis. These GMS strains induce mucosal, systemic, and cellular immune responses in mice against viral, bacterial, and parasite pathogens. They are also designed for anti-cancer material delivery. Salmonella has also been extensively explored as a potential cancer treatment. Many tumors release 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. However, the induction of strong innate and adaptive immune responses against bacterial carriers may misdirect the host's immune response to bacteria and therefore disrupt theeliciting of protective immunity against targeted pathogens and cancers, which limits the performance of the GMS delivery system for use as a universal vaccine and therapeutic delivery platform. Therefore, strategies to prevent host immune response to vaccines and therapeutics delivering GMS strains are needed.SUMMARY OF THE DISCLOSURE

[0005] Provided herein is a genetically modified Salmonella bacterium comprising a deletion of one or more genes selected from yfgL, yiaD, STM1540, STM1940, slyB,fepA, iroN, cirA, ydcW, and yfiO.

[0006] The bacterium may further comprise a deletion of pmi. The bacterium may further comprise at least one of the mutationsThe bacterium may further comprise the mutation

[0007] In embodiments, the bacterium comprises the following mutations:

[0008] In embodiments, the bacterium comprises the following mutations:

[0009] In embodiments, the bacterium comprises the following mutations:

[0010] In embodiments, the bacterium comprises the following mutations:

[0011] In embodiments, the bacterium comprises the following mutations

[0012] In embodiments, the bacterium comprises the following mutations:

[0013] The bacterium may further comprise a recombinant polynucleotide encoding arabinose- regulated murA and asdA.

[0014] The bacterium may comprise a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 41.

[0015] The bacterium may further comprise a recombinant polynucleotide encoding TRAIL.

[0016] The bacterium may comprise a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 42.

[0017] The bacterium further comprises a recombinant polynucleotide encoding a DNA nuclear targeting sequence (DTS). The DTS may be an SV40 enhancer. The DTS may be a single copy of an SV40 72 bp enhancer element, and an SV40 late poly A. The recombinant polynucleotide may further encode a CMV immediate-early gene enhancer / promoter. The recombinant polynucleotide may further comprise a binding site for at least one of NF-κB and AP-2.

[0018] The bacterium may be S. Typhimurium. The bacterium may be 5. Typhimurium UK-1.

[0019] The bacterium may further comprise a therapeutic. The therapeutic may be an anti-viral vaccine.

[0020] A method for treating a subject in need thereof is also provided, the method comprising administering a genetically modified Salmonella bacterium described herein. Administering may comprise oral administration, intravesical injection, subcutaneous injection, or intra-tumoral injection of the bacterium into the subject.DETAILED DESCRIPTION

[0021] The inventors have developed a self-destructive recombinant attenuated Salmonella vaccine and therapeutic delivery system. These genetically modified Salmonella strains allow the release of protective antigens and therapeutics expressed in Salmonella cells and / or DNA vaccine vectors encoding targeted proteins after programmed cell lysis in vivo, which could stimulateprotective immunities against a diversity of pathogens and release anticancer material. Bacterialbased vaccine vectors remain promising vaccine and therapeutic delivery platforms, however, the induction of strong innate and adaptive immune responses against bacterial vectors may misdirect the host's immune response to bacteria and subsequently disrupt the eliciting of protective immunity against targeted diseases.

[0022] To develop a highly efficient antigen / DNA vaccine and therapeutic delivery platform against pathogens and cancers, the inventors have redecorated the Salmonella cell surface for shifting the host immune response to target pathogens and cancers and avoid the potential double burden of immune responses to both Salmonella carriers and targeted diseases. These GMS constructions can be rapidly modified to synthesize and deliver new protective antigens / DNA vaccines, therapeutics and allow them to be rapidly manufactured as thermo-stable vaccines and therapeutics, in a cost-effective manner.

[0023] In a first aspect, provided herein is a genetically modified Salmonella (GMS) bacterium comprising a deletion of one or more genes selected from yfgL. (encoding outer membrane protein assembly factor BamB), yiaD (encoding outer membrane protein A), STM1540 (encoding hydrolase), STM 1940 (encoding cell wall-associated hydrolase), slyB (encoding outer membrane lipoprotein SlyB), fepA (encoding outer membrane ferrienterobactin receptor fepA), iroN (encoding outer membrane siderophore receptor), cirA (encoding catecholate siderophore receptor), ydc W (encoding an aldehyde dehydrogenase), and yfiO (also known as OmpX, encoding an outer membrane protein assembly complex). The proteins encoded by yfgL, yiaD, STM1540, STM 1940, slyB, fepA, iroN, cirA, yfiO are located at the outer membrane. The aldehyde dehydrogenase encoded by ydcW is a periplasmic protein. Deletion of one or more of the genes reduces the immunogenicity of the bacterium and prevent or reduce the bacterium being targeted by a host in which the bacterium has been introduced. In embodiments, the genetically modified bacterium comprises a deletion of all of yfgL, yiaD, STM1540, STM1940, slyB, fepA, iroN, cirA, ydcW, and yfiO.

[0024] 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, orSalmonella Dublin. As used herein, “S. Typhimurium” refers to an isolate of A 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.

[0025] 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 an essential nucleic acid under the control of a regulatable promoter, such that the nucleic acid is not expressed in a host.

[0026] The GMS bacterium may further comprise a deletion of pmi (encoding mannose-6- phosphate isomerase).

[0027] The GMS bacterium may further comprise at least one of the mutationswhere P stands for promoter, TT for transcription terminator. The GMS bacterium may comprise bothmutations. The “Δ” 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.

[0028] The GMS bacterium may comprise pYA3681. The bacterium may comprise a vector comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 41.

[0029] The GMS bacterium may further comprise the mutation

[0030] In an embodiment, the GMS bacterium comprises the following mutations:. In an embodiment, the GMS bacterium comprises the following mutations:. In an embodiment, the GMS bacterium comprises the following mutations:. In an embodiment, the GMS bacterium comprises the following mutations. In an embodiment, theGMS bacterium comprises the following mutations. In an embodiment, the GMS bacterium comprises the followingmutations:

[0031] The GMS bacterium may further comprise a recombinant polynucleotide encoding TNF- related apoptosis-inducing ligand (TRAIL).

[0032] The GMS bacterium may further comprise a recombinant polynucleotide encoding arabinose-regulated murA and asdA. 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. Lysisalso 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 PBAD murA 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 PBAD murA 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.

[0033] 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

[0034] 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. 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 amutation, which initially prevents breakdown of accumulated arabinose at the time of inoculation.

[0035] Bacteria that comprise 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 GTGto decrease translation efficiency) under the control of an araC PBAD promoter. 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 GTGto 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 PR is 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 PR directed synthesis of anti-sense mRNA to further block translation of asdA and murA mRNA. The araC PBAD sequence is also not from E. coll B / r as originally described but represents a sequence derived from E. coli K-12 strain 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. coll 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.

[0036] 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.

[0037] 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 regulatorysystems 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 iSrelA that uncouples cell wall-less death from dependence on protein synthesis.

[0038] Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterob acteriaceae. 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.

[0039] A recombinant bacterium may also comprise a ΔreM::TT araC PBAD lacI 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 Ptre. 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.

[0040] 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: :PIppIpxE mutation.

[0041] Moreover, a bacterium comprising a nucleic acid vaccine vector may also comprise a mutation that eliminates the periplasmic endonuclease I enzyme, such as a EendA mutation. This type of mutation is designed to increase vector survival upon the vector's release into the host cell.

[0042] The GMS bacterium may further comprise elements for improving vector import into the nuclei of host cells. For example, the GMS bacterium may further comprise a recombinant polynucleotide encoding a single copy of an SV40 72 bp enhancer element, (which serves as a DNA nuclear targeting sequence (DTS)), and an SV40 late poly A. The GMS bacterium may further comprise a recombinant polynucleotide may further encode a CMV immediate-early gene enhancer / promoter (CMV E / P). The GMS bacterium may further comprise a recombinantpolynucleotide may further comprise at least one of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) binding site and an AP-2 adaptor binding site, each of which serve as a DTS. Use of the described DTS’s in DNA vaccine vectors for Salmonella are described in Kong et al. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform. Proc Natl Acad Sci U S A. 2012 Nov 20; 109(47): 19414-9, which is incorporated by reference herein in its entirety.

[0043] The GMS bacterium may be S. Typhimurium. The bacterium may be the strain S. Typhimurium UK-1.

[0044] The GMS bacterium may further comprise a therapeutic.

[0045] 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 described herein. 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.

[0046] 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.

[0047] 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 asubject in need thereof comprises 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.

[0048] 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 depends upon the host receiving the modified bacteria as well as factors such as the size, weight, and age of the host.

[0049] 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).

[0050] 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.

[0051] 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 or condition 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.

[0052] As used herein, the term “therapeutic” refers to an agent used to treat or prevent a disease. The term “therapeutic” encompasses vaccines. The term “vaccine” refers to an agent used to prevent or ameliorate the effects of a future infection by a pathogen.

[0053] 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.

[0054] 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 strain. In other cases, administering comprises intra-tumoralinjection of the genetically modified Salmonella strain. The mode of administration can be determined based on the physical location, type, or the number of tumors in the subject's body.

[0055] Clinicians, physicians, and other health care professionals can administer genetically modified Salmonella strains 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, 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.

[0056] 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 strains 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.

[0057] 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.

[0058] 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 some embodiments, “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 deoxy cytidine); 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, 0(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).

[0059] 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 usually associated whether it is derived from a naturally occurring source or made synthetically, in whole or in part.

[0060] 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 is 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.

[0061] Nucleic acids, proteins, and / or other moi eties 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.

[0062] 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 disclosuredescribed 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.

[0063] So that the compositions and methods provided herein may more readily be understood, certain terms are defined:

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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 usedherein 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.

[0068] 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.EXAMPLES

[0069] The following examples will enable one of skill in the art to more readily understand the principles thereof. The following examples are presented by way of illustration and are not meant to be limiting in any way.

[0070] Example 1

[0071] Available means to improve genetically engineer recombinant Salmonella strains for redecorate the cell surface of the GMS strains are as follows.

[0072] i. Genetically altering S. enterica serotypes to display regulated delayed attenuation. We have developed multiple means to achieve regulated delayed attenuation in Salmonella strains. Some strategies involve a smooth-to-rough phenotypic change in LPS in vivo using strains with the Spmi mutation (A stands for deletion) that lack phosphomannose isomerase needed to interconvert fructose-6-phosphate and mannose-6-phosphate. Growth of such strains in media with mannose results in synthesis of complete LPS O-antigen with the ability of the strain, after oral immunization, to colonize lymphoid tissues, but loss of the LPS O-antigen side chains after about seven cell divisions in vivo since nonphosphorylated mannose is unavailable.

[0073] ii. Construction of programmed lysis GMS strains that enable regulated delayed lysis in vivo to confer attenuation and complete biological containment. A means to achieve regulated delayed attenuation is a host-vector system that regulates a delayed lysis in vivo. This depends on the chromosomalmutations (P stands for promoter, TT for transcription terminator) that impose requirements for the peptidoglycan-essential diaminopimelic acid (DAP) and muramic acid constituents coupled with a plasmid vector in which expression of the murA and asdA genes are also subject to arabinose-dependent araC PBAD control. Collectively, these attributes diminish the ability of GMS strains to persist / survive in vivo. These Salmonella host-vector systems are ideal for delivery of antigens that are difficult to secrete due to structural attributes and / or DNA vaccine vectors.

[0074] iii. Construction of hyper-invasive Salmonella strain to increase the efficient for therapeutic delivery. One of the major mechanisms of 5. Typhimurium invasion of animal hosts is by entering and traversing the epithelial monolayer through microfold (M) cells.The hilA (hyper-invasion locus) regulator encodes an OmpR / ToxR family transcriptional regulator that activates expression of invasion genes in response to both environmental and genetic regulatory factor. To improve M cell-mediated Salmonella invasion, we replaced the hilA promoter with the Ptrc promoter to enable constitutive synthesis of HilA. The S. Typhimurium strain withmodification invaded human intestinal Int-407 cells and colonized mouse tissues in significantly greater numbers than the wild-type strain.

[0075] iv. Construction of tumor navigating Salmonella strains to increase the efficient for therapeutic delivery. Salmonella has been extensively explored as a potential cancer treatment [14- 23], Many tumors release 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, we reprogrammed the chemotaxis system resulting in strains GMS515(for DNA vaccine delivery).The growing understanding of the mechanisms that regulate programmed cell death has led to the emergence of new agents capable of restarting apoptosis in malignant cells. The extrinsic apoptosis pathway is initiated via death receptor activation by their cognate ligand, TRAIL. We have successfully delivered TRAIL using their newly developed tumor-targeting self-eradicating GMS strains that can actively search for tumors (both primary and metastatic lesions), colonize thetumor, and eventually release cancer-killing material in the tumor bed, resulting in tumor cell killing, antigen release, innate immune cell attraction / activation and potent induction of anti-tumor immunity. The reprogrammed chemotaxis system endues GMS strains with their superior ability of tumor homing, cancer cell seeking, cellular attachment as well as invasion.

[0076] v. Construction of lysis vectors for antigen delivery. To avoid the use of plasmid vectors with non-permitted drug resistance genes and to stabilize plasmid vectors in recombinant attenuated Salmonella strains in vivo, we developed a balanced-lethal Salmonella host-vector system with deletion of the asdA gene to impose an obligate requirement for diaminopimelic acid (DAP) and a plasmid vector with the wild-type asdA gene. To develop a regulated lysis system, we converted Salmonella strains possessing the mutations described in the preceding two sections into balanced-lethal vector-host constructions by using plasmid vectors that possess the wild-type murA and asdA genes. The plasmid component is a pBR ori-containing pYA3681, which encodes arabinose-regulated murA and asdA expression. Upon invasion of arabinose-free host tissues, transcription of asdA and murA ceases and levels of their gene products decrease due to cell division. These Salmonella host-vector systems are ideal for delivery of antigens that are difficult to secrete due to structural attributes or DNA vaccine vectors. In addition, they provide complete biological containment with no persistence in vivo and no survival if excreted.

[0077] vi. Construction of lysis vectors for DNA vaccine delivery. Non-viral DNA vaccine vector nuclear import 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 its function is a single copy of a 72-bp element of the SV40 enhancer. In addition, the synthesis of eukaryotic transcription factors, e.g., NF-κB and AP-2, are stimulated by Salmonella infection. These transcription factors can bind to nonviral DNA vaccine vectors, allowing the nuclear locating signal to mediate import of plasmid DNA into the nucleus. Nuclease degradation of DNA vaccine vectors after delivery and during trafficking to the nucleus is another barrier that leads to inefficient DNA vaccination. Use of the SV40 late poly A has improved resistance to attack from mammalian nucleases. Therefore, the 72 bp DTS (I) of the SV40 enhancer and the SV40 late poly A were included in an improved DNA vaccine vector pYA4545. We also designed artificial DNA binding sites DTS (II) for NF-KB and AP-2 and inserted them in upstream of the CMV E / P in pYA4545 as an additional DNA nuclear targeting and enhancer sequence.

[0078] The invention is to redecorate the cell surface of the GMS strains by eliminating selected immunodominant antigens to weaken / impair the host immune responses against Salmonella-based therapeutic delivery system. The properties, such as high in vivo abundance or immunodominance, of many Salmonella surface exposed and internal antigens have been identified in convalescent individuals and confirmed in a mouse model that closely mimics human typhoid fever. We remove selected immunodominant antigens, including surface exposed antigens and internal antigens, from our GMS strains for therapeutic delivery.

[0079] We have deleted the genes encoding the selected immunodominant antigens from GMS strains and select desired genetic attributes. The genes encoding the following selected immunodominant surface and internal antigens are deleted from the chromosome of wild-type S. Typhimurium UK-1 strain: yfgL (lipoprotein, located in the outer membrane, namely OM), yiaD, (lipoprotein, OM), STM1540 (lipoprotein, OM), STM1940 (lipoprotein, OM), slyB (lipoprotein, OM), fepA (OM), iroN (OM), cirA (OM), ydcW (periplasmic protein), and yfiO (OM), respectively. The resulting strains are examined regarding their growth and colonization abilities, as well as the ability to persist in Peyer’s patch, spleen, and liver of immunized BALB / c mice at days 3, 7, 11, 15, 19, 23, 27, 31, and 35 respectively. The reduced immune responses against Salmonella strains is also confirmed by measuring the serum IgG against Salmonella at weeks 4, 6, 8, and 12 after immunization. The genetic attributes, which reduce the desired immune responses to Salmonella but retain the growth rate as well as colonization and persistent abilities, are introduced to the GMS strains.

[0080] The genotype of strains are:

[0081] GMS5008:

[0083] GMS5010:

[0084] GMS501 1 :

[0085] GMS5012:

[0087] Suicide vector technology was used to generate deletion and insertion mutations. Primers used for construction of the suicide vectors are shown at Table 1.

[0088] Table 1. Primers used for construction of suicide vectors; and empty suicide vector

[0089] 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.

[0090] EMBODIMENTS

[0091] Embodiment 1. A genetically modified Salmonella bacterium comprising a deletion of one or more genes selected from yfgL, yiaD, STM1540, STM1940, slyB, fepA, iroN, cirA, ydcW, and yfio.

[0092] Embodiment 2. The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium further comprises at least one of the mutationsand.

[0093] Embodiment 3. The genetically modified Salmonella bacterium of embodiment 2, wherein the bacterium further comprises a deletion of pmi.

[0094] Embodiment 4. The genetically modified Salmonella bacterium of embodiment 2 or 3, wherein the bacterium further comprises the mutation.

[0095] Embodiment 5. The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium comprises the following mutations:

[0096] Embodiment 6. The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium comprises the following mutations:

[0097] Embodiment 7. The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium comprises the following mutations:

[0098] Embodiment 8 The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium comprises the following mutations:

[0099] Embodiment 9. The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium comprises the following mutations:

[0100] Embodiment 10. The genetically modified Salmonella bacterium of embodiment 1, wherein the bacterium comprises the following mutations:

[0101] Embodiment 11. The genetically modified Salmonella bacterium of any one of embodiments 1-10, wherein the bacterium further comprises a recombinant polynucleotide encoding arabinose-regulated murA and asdA.

[0102] Embodiment 12. The bacterium of any one of embodiments 1-11, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 41.

[0103] Embodiment 13. The genetically modified Salmonella bacterium of any one of embodiments 1-12, wherein the bacterium further comprises a recombinant polynucleotide encoding TRAIL.

[0104] Embodiment 14. The bacterium of any one of embodiments 1-11, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 42.

[0105] Embodiment 15. The genetically modified Salmonella bacterium of any one of embodiments 1-11, wherein the bacterium further comprises a recombinant polynucleotide encoding a DNA nuclear targeting sequence (DTS).

[0106] Embodiment 16. The genetically modified Salmonella bacterium of embodiment 15, wherein the DTS is an SV40 enhancer.

[0107] Embodiment 17. The genetically modified Salmonella bacterium of embodiment16, wherein the DTS is a single copy of an SV40 72 bp enhancer element, and an SV40 late poly A.

[0108] Embodiment 18. The genetically modified Salmonella bacterium of embodiment17, wherein the recombinant polynucleotide further encodes a CMV immediate-early gene enhancer / promoter.

[0109] Embodiment 19. The genetically modified Salmonella bacterium of any one of embodiments 1-18, wherein the recombinant polynucleotide further comprises a binding site for at least one of NF-KB and AP-2.

[0110] Embodiment 20. The genetically modified Salmonella bacterium of any one of embodiments 1-19, wherein the bacterium is S. Typhimurium.

[0111] Embodiment 21. The genetically modified Salmonella bacterium of embodiment 20, wherein the bacterium is S. Typhimurium UK-1.

[0112] Embodiment 22. The genetically modified Salmonella bacterium of any one of embodiments 1-21, wherein the bacterium further comprises a therapeutic.

[0113] Embodiment 23. The genetically modified Salmonella bacterium of embodiment 22, wherein the therapeutic is an anti-viral vaccine.

[0114] Embodiment 24. A method for treating a subject in need thereof, the method comprising administering a genetically modified Salmonella bacterium of embodiment 23.

[0115] Embodiment 25. The method of embodiment 24, wherein administering comprises oral administration, intravesical injection, subcutaneous injection, or intra-tumoral injection of the bacterium into the subject.

Claims

CLAIMSWe claim:

1. A genetically modified Salmonella bacterium comprising a deletion of one or more genes selected from yfgL, yiaD, STM1540, STM1940, slyB, fepA, iroN, cirA, ydcW, andyfiO.

2. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium further comprises at least one of the mutations.

3. The genetically modified Salmonella bacterium of claim 2, wherein the bacterium further comprises a deletion of pmi.

4. The genetically modified Salmonella bacterium of claim 2, wherein the bacterium further comprises the mutation5. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium comprises the following mutations:

6. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium comprises the following mutations:

7. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium comprises the following mutations:

8. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium comprises the following mutations:

9. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium comprises the following mutations:

10. The genetically modified Salmonella bacterium of claim 1, wherein the bacterium comprises the following mutations:

11. The genetically modified Salmonella bacterium of any one of claims 1-10, wherein the bacterium further comprises a recombinant polynucleotide encoding arabinose-regulated murA and asdA .

12. The genetically modified Salmonella bacterium of any one of claims 1-11, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 41.

13. The genetically modified Salmonella bacterium of any one of claims 1-12, wherein the bacterium further comprises a recombinant polynucleotide encoding TRAIL.

14. The genetically modified Salmonella bacterium of any one of claims 1-11, wherein the bacterium comprises a lysis vector comprising a sequence having at least 90% identity to SEQ ID NO: 42.

15. The genetically modified Salmonella bacterium of any one of claims 1-11 , wherein the bacterium further comprises a recombinant polynucleotide encoding a DNA nuclear targeting sequence (DTS).

16. The genetically modified Salmonella bacterium of claim 15, wherein the DTS is an SV40 enhancer.

17. The genetically modified Salmonella bacterium of claim 16, wherein the DTS is a single copy of an SV40 72 bp enhancer element, and an SV40 late poly A.

18. The genetically modified Salmonella bacterium of claim 17, wherein the recombinant polynucleotide further encodes a CMV immediate-early gene enhancer / promoter.

19. The genetically modified Salmonella bacterium of any one of claims 1-18, wherein the recombinant polynucleotide further comprises a binding site for at least one of NF-KB and AP-2.

20. The genetically modified Salmonella bacterium of any one of claims 1-19, wherein the bacterium is S. Typhimurium.

21. The genetically modified Salmonella bacterium of claim 20, wherein the bacterium is S. Typhimurium UK-1.

22. The genetically modified Salmonella bacterium of any one of claims 1-21, wherein the bacterium further comprises a therapeutic.

23. The genetically modified Salmonella bacterium of claim 22, wherein the therapeutic is an anti-viral vaccine.

24. A method for treating a subject in need thereof, the method comprising administering a genetically modified Salmonella bacterium of claim 23.

25. The method of claim 24, wherein administering comprises oral administration, intravesical injection, subcutaneous injection, or intra-tumoral injection of the bacterium into the subject.

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