RECOMBINANT THERAPEUTIC INTERVENTIONS FOR CANCER

Abstract

Described are compositions and methods for treating or preventing cancer in a subject by administering a pharmaceutical composition comprising a strain of Mycobacteria including an expression vector of the present invention into the bladder of a subject. The pharmaceutical composition may be administered by any suitable means including by a catheter.

Claims

1. A vector comprising a nucleic acid sequence expressing a protein or functional part thereof that makes a STING agonist.

2. The vector of claim 1 wherein the STING agonist is selected from the group consisting of 3-5 c-di-AMP (also known as c-di-AMP); 3-5 c-di-GMP (also known as c-di-GMP); 3-3cGAMP ; 2-3cGAMP and a combination thereof.

3. The vector of claim 2 comprising the nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3-3 cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a 2-3 cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (disA) protein, or a functional part thereof and a combination thereof.

4. The vector of claim 1 further comprising a fifth nucleic acid sequence encoding a PanC and a PanD protein or functional part thereof.

5. The vector of claim 4 wherein the vector is free of an antibiotic resistance gene.

6. The vector of claim 1 wherein the vector is selected from the group consisting of a vector that stably integrates into the genome of a bacterium, a vector that stably replicates episomally in multiple copies within a bacterium, and/or a combination thereof.

7. The vector of claim 3 further comprising a fifth nucleic acid sequence that encodes a protein or nucleic acid sequence that knocks out the expression of a phosphodiesterase gene or a phosphodiesterase domain of a Mycobacterium.

8. The vector of claim 3 wherein the third nucleic acid sequence overexpresses the cyclase domains of the cyclic GMP-AMP synthase (cGAS) protein.

9. The vector of claim 3 wherein the third nucleic acid sequence expresses a cyclic GMP-AMP synthase (cGAS) protein having a regulatory DNA recognition capability that is non-functional.

10. A strain of Mycobacteria comprising a vector comprising a protein or functional part thereof that makes a STING agonist.

11. The strain of claim 10 wherein the STING agonist is selected from the group consisting of 3-5 c-di-AMP (also known as c-di-AMP); 3-5 c-di-GMP (also known as c-di-GMP); 3 -3cGAMP; 2 -3 cGAMP and a combination thereof.

12. The strain of claim 10 wherein the a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof a second nucleic acid sequence encoding a 3-3 cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof a third nucleic acid sequence encoding a 2-3 cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof a fourth nucleic acid sequence encoding a DNA integrity scanning (DisA) protein, or a functional part thereof and a combination thereof.

13. The strain of claim 12 wherein the strain of Mycobacteria is Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof.

14. The strain of claim 13 wherein the strain of Mycobacteria is Mycobacterium bacillus Calmette Guerin (BCG).

15. The strain of claim 14 wherein the strain is a panthothenate (panCD mutant) auxotroph of BCG and the vector comprises a panCD nucleic acid encoding a PanC protein or a functional part thereof and a nucleic acid sequence encoding a PanD protein or functional part thereof.

16. The strain of claim 16 wherein the strain is free of a genomic antibiotic resistance gene.

17. The strain of claim 16, wherein the vector is free of an antibiotic resistance gene.

18. The strain of claim 10, wherein the vector is selected from a group consisting of a vector that is integrated into the Mycobacterium chromosome, stably replicates episomally in multiple copies with the Mycobacterium, and a combination thereof.

19. The strain of claim 10 wherein the strain is free of an antibiotic resistance gene.

20. A pharmaceutical composition, comprising any one of the strains of Mycobacteria of claims 10, and (ii) a pharmaceutically acceptable carrier.

21. A method of of eliciting a Type 1 interferon response, enhancing the expression of pro-inflammatory cytokine, and/or eliciting trained immunity in subject comprising the steps of: administering a pharmaceutical composition comprising anyone of the strains strains of Mycobacteria of claims 10, and eliciting a Type 1 interferon response, enhancing the expression of pro-inflammatory cytokine, and/or eliciting trained immunity in the subject.

22. The method of claim 21, wherein the pharmaceutical composition is administered into the bladder of the subject by a catheter.

23. A method of using a strain of Mycobacteria comprising a vector expressing a protein that makes a STING agonist to treat or prevent cancer in a subject comprising the steps of: administering a pharmaceutical composition comprising a strain of Mycobacteria comprising a vector expressing a protein that makes a STING agonist or a functional part thereof to a subject having cancer; and treating or preventing cancer in the subject.

24. The method of claim 23 wherein the STING agonist is selected from the group consisting of 3-5 c-di-AMP (also known as c-di-AMP); 3-5 c-di-GMP (also known as c-diGMP); 3-3cGAMP; 2-3cGAMP and a combination thereof.

25. The method of claim 24 comprising a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3-3cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a 2-3 cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (DisA) protein, or a functional part thereof and a combination thereof.

26. The method of claim 23 wherein the cancer is selected from the group consisting of epithelial cancers, breast cancer, non-muscle invasive bladder cancer, and a combination thereof.

27. The method of claim 26 wherein the cancer is non-muscle invasive bladder cancer and is a BCG-unresponsive non-muscle invasive bladder cancer (BCG-unresponsive NMIBC) and the pharmaceutical composition is administered by intravesical instillation.

28. The method of claim 26 wherein the cancer is non-muscle invasive bladder cancer and is a BCG-nave non-muscle invasive bladder cancer (BCG-nave NMIBC) and the pharmaceutical composition is administered by intravesical instillation.

29. The method of claim 26 wherein the epithelial cancer is selected from the group consisting of colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngeal cancer, endobronchial cancer, and a combination thereof and the pharmaceutical composition is administered by to a luminal surface of the epithelial cancer.

30. The method of claim 23 wherein the cancer is selected from a solid tumor, liquid tumor.

31. The method of claim 30 wherein the pharmaceutical composition is administered by intratumoral injection.

32. The method of claim 30 wherein the pharmaceutical composition is administered by systemic infusion.

33. The method of claim 23 comprising the step of administering a checkpoint inhibitor.

34. The method of claim 33 wherein the checkpoint inhibitor is selected from the group consisting of ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), cemiplimab (anti-PD-1), atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1) and a combination thereof.

35. The method of claim 23 wherein the cancer is bladder cancer and a catheter administers the pharmaceutical composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1A-1B Mycobacteria overexpressing disA from the pSD5B P.sub.hsp60::disA plasmid construct release large amounts of c-di-AMP into the macrophage cytosol and transcribe high levels of disA mRNA. A. J774 macrophages infected with M.tb harboring the pSD5B P.sub.hsp60::disA plasmid or wild type M.tb (CDC1551) at an MOI of 1:20. Intramacrophage levels of c-di-AMP were determined by LC-MS/MS after 24 hours of infection. As can be seen, the M.tb-disA-OE strain produces 15-fold more c-di-AMP than wild type M.tb (CDC1551). The BCG-disA-OE would be expected to show similarly high levels of c-di-AMP. (Data are from Dey B, Dey R J, Cheung L S, Pokkali S, Guo H, Lee J H, Bishai W R. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis. Nat Med. 2015; 21:401-6. PMID: 25730264.) B. BCG-Pasteur harboring the pSD5B P.sub.hsp60::disA plasmid or BCG-Pasteur-WT were grown to mid-exponential phase. Bacteria were lysed and mRNA was prepared. The levels of disA mRNA were determined by quantitative RT-PCR. The BCG-disA-OE strain produces 50-fold more disA mRNA than BCG-Pasteur-WT.

[0076] FIG. 2. BCG overexpressing disA augments pro-inflammatory cytokines. Gene expression profiling (qPCR) of pro-inflammatory cytokines and IFN- in mouse BMDMs challenged with wild-type and disA overexpression strains of BCG-Pasteur.

[0077] FIG. 3. BCG overexpressing disA augments IRF3 signaling. Effect of disA overexpression on activation of IRF pathway measured by IRF-SEAP QUANTI Blue reporter assay. The culture supernatants of infected RAW-Blue ISG cells were assayed for IRF activation. The image below the IRF-activation graph represents QUANTI Blue assay plate and sample wells; treatment parameters for column of wells correspond to those defined for the bars above aligned with the wells. BCG-disA-OE in this figure is derived from BCG Pasteur.

[0078] FIG. 4A-4C. Increased pro-inflammatory cytokines in response to disA overexpression. Differential expression of TNF- (A), IL-6 (B) and IL-1 (C) in mouse BMDMs challenged with wild-type and disA overexpression strains of BCG-Pasteur. Culture supernatants were assayed by ELISA for different cytokines.

[0079] FIG. 5. BCG overexpressing disA induces differential immune response in human bladder cancer cells (RT4). Differential gene expression in human RT4 bladder cancer cells challenged with wild-type BCG-Pasteur, wild-type BCG-Tice strain, and BCG-Pasteur-disA-OE Expression levels of mRNA was measured using a SYBR green-based quantitative real-time PCR.

[0080] FIG. 6. Schematic workflow of testing relative therapeutic efficacy of wild-type and BCG-disA-OE strains.

[0081] FIG. 7. Tumor involvement index of tumor-bearing rats untreated or treated with WT BCG or rBCG overexpressing disA (rBCG=BCG-Pasteur-disA-OE; wtBCG=BCG-Pasteur).

[0082] FIG. 8. Immune profiling of MNU-induced Fisher rat urinary bladder tumors in response to intravesical therapy using different strains of BCG. Differential gene expression in Rat bladder tumor cells after therapy with wild-type and disA overexpression strains of Mycobacterium bovis BCG-Pasteur. Expression levels of mRNA were measured using a TaqMan-based quantitative real-time PCR. BCG-WT is BCG Pasteur and BCG-disA-OE was derived from BCG Pasteur in this figure.

[0083] FIG. 9. Gene expression profiling of bladders from MNU tumor bearing rats untreated or treated with WT or rBCG overexpressing disA.

[0084] FIG. 10. Summary of relative gene expression by BCG-disA-OE versus BCG-WT in different cells or tissues. Mouse bone marrow-derived macrophages (BMDM), human immortalized bladder cancer cell lines RT4 and 5637, and rat immortalized bladder cancer cell lines were infected with BCG-disA-OE and BCG-WT for 24 hours and mRNA was prepared from the cells. Rats were exposed to MNU by intravesical instillation over 8 weeks and then treated with either BCG-disA-OE or BCG-WT by intravesical instillation for 8 weeks. Bladders were removed upon necropsy at week 16, and mRNA was prepared. Quantitative RT-PCR for the cytokine or chemokine genes indicated was performed. The changes shown are the fold-induction or reduction observed with BCG-disA-OE normalized to that seen with BCG-WT. BCG-WT is BCG Pasteur and BCG-disA-OE was derived from BCG Pasteur in this figure.

[0085] FIG. 11. Proposed mechanisms of action of BCG overexpressing disA.

[0086] FIG. 12. Molecular genetic modifications to BCG which will increase the levels of CDN PAMP and DAMP molecules which are STING agonists. PAMP: pathogen associated molecular pattern (made by bacteria): c-di-AMP (disA), c-di-GMP (Rv1354c), 33-cGAMP (dncV). DAMP: danger-associated molecular pattern (made by host): 2-3-cGAMP. OE: overexpressor. KO: knockout (gene replacement).

[0087] FIG. 13. Diagram of two cyclic dinucleotide cyclase and phosphodiesterase proteins present in BCG: BCG_RS07340 and BCG AHM07112. BCG_RS07340 is a bifunctional protein with both CDN cyclase and CDN PDE activities. BCG AHM07112 is a CDN PDE. The domains are: GAF (regulatory), GGDEF (diguanylate cyclase), and EAL diguanylate phosphodiesterase.

[0088] FIG. 14. M tuberculosis harboring the pSD5B Phs.sub.p6o::disA plasmid (M.tb-disA-OE or M.tb-OE) is significantly attenuated for virulence in mice compared to wild type M.tb (M.tb-CDC1551). 6-7-week-old female BALB/c mice (n=10 per group) were infected as described above with 3.5 log.sub.10 CFU by aerosol infection. Day 1 CFU counts were performed on 3 mice in each group and confirmed the implantation of 3.5 log10 CFU units. Mice were held until death. As can be seen, the median time to death for wild-type M. tuberculosis infection was 150.5 days. In contrast, mice infected with the same inoculum of M.tb-disA-OE (M.tb-OE) had a median time to death of 321.5 days (p<0.001). The BCG-disA-OE is expected to show similar loss of virulence in mice compared with BCG-WT. (Data are from Dey B, Dey R J, Cheung L S, Pokkali S, Guo H, Lee J H, and Bishai W R. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis. Nat Med. 2015; 21: 401-6. PMID: 25730264.)

[0089] FIG. 15A-15B. Other BCG strains are also active: BCG Tice strain overexpressing disA also shows induction of proinflammatory cytokines similar to BCG Pasteur overexpressing disA. Bone marrow derived macrophages were challenged with wild-type and disA overexpressing strains of both BCG Pasteur and BCG Tice strains at an M.O.I of 1:20 for 15 h. Culture supernatants were harvested and probed for cytokines using ELISA. Differential expression pattern of TNF- (A) and IL-6 (B) in mouse BMDM challenged with the two different strains of BCG. The BCG-Tice strain was from the commercially available Onco-Tice product.

[0090] FIG. 16. The Type I interferon responses in macrophages in response to BCG-disA-OE are STING-dependent. Bone marrow-derived macrophages from STING-ablated (KO) and control mouse were challenged with wild-type and disA OE strains of BCG Pasteur for 24 h. Culture supernatants were probed for IFN- levels using ELISA.

[0091] FIG. 17 Shows that intravesical instillation of BCG-disA-OE displays greatest antitumor efficacy (statistically significant improvement in pathology) in the MNU carcinogen model of non-muscle invasive bladder cancer (NIMBC). Groups of rats received 4 intravesical treatments with MNU over the first 8 weeks (one treatment every 2 weeks) to elicit NIMBC. Over the next 8 weeks they received 4 intravesical treatments with either PBS (untreated), BCG-WT, or BCG-disA-OE (one treatment every 2 weeks). At the end of the 16-week experiment, rats were sacrificed, and their bladders were removed. A portion of the bladder was fixed and subjected to H&E staining and then interpreted in a blinded fashion by a Board-certified urologic pathologist. The tumor involvement score and cancer stage (T2-3, T1, CIS+papillary lesions, CIS alone, or normal-dysplastic) were determined and are shown. As may be seen BCG-disA-OE instillation resulted into statistically significantly and lower tumor involvement index than PBS (untreated) while BCG-WT was not statistically significantly superior to PBS. This 16 -week experiment was performed twice. The data in FIG. 7 represent the results of Experiment 1. The data in this figure (FIG. 17) represent the combined results of Expt 1 plus Expt 2. The qPCR data shown in FIG. 8 and FIG. 9 were obtained using bladder tissue at necropsy from the end of Expt 1.

[0092] FIG. 18 shows that BCG-disA-OE reduces Tregs (CD4.sup.+CD25.sup.+Foxp3.sup.+) in murine syngeneic bladder cancer tumors. Mice were implanted on the flank with 510.sup.6 BBN975 murine bladder cancer tumor cells. When the tumors were 1.5 cm in diameter, mice received 3 intratumoral injections of either PBS (control), BCG-WT, or BCG-disA-OE (one treatment every 2 days). Two days after the last intratumoral treatment, mice were sacrificed and their spleens and tumors were removed. After tumor cell dispersal, the cell preparations were stained and subjected to flow cytometry. As may be seen BCG-disA-OE led to reduced tumor CD4.sup.+ Tregs, reduced tumor CD8.sup.+ Tregs, and reduced spleen CD4.sup.+ Tregs.

[0093] FIG. 19 shows that BCG-disA-OE is safer than BCG-WT in two mouse models. In Panel A, groups of BALB/c mice (immunocompetent) were exposed to 110.sup.3 CFU (confirmed by sacrificing a group of mice and determining day 1 lung CFU counts) of either BCG-WT or BCG-disA-OE using a Glas-Col aerosolization chamber. After 4 weeks, the mice were sacrificed from each group, their lungs were removed, homogenized, and plated on 7H11 agar plates. The figure shows the mean CFU counts for the BCG-WT and BCG-disA-OE-infected mouse lungs. As may be seen a statistically significantly lower lung CFU burden was observed with BCG-disA-OE compared with BCG-WT. In Panel B groups of SCID mice (immunosuppressed) were exposed to 110.sup.2 CFU (confirmed by sacrificing a group of mice and determining day 1 lung CFU counts) of either BCG-WT or BCG-disA-OE using a Glas-Col aerosolization chamber. A third group was uninfected. The figure shows a Kaplan-Meier survival curve for the groups of mice. As may be seen BCG-disA-OE-infected mice had a statistically significantly longer survival time than BCG-WT-infected mice.

[0094] FIG. 20 shows that BCG-disA-OE elicits statistically significantly higher levels of Trained Immunity immunological and epigenetic marks in CD14.sup.+ human monocytes than does BCG-WT. Trained Immunity refers to the ability of a first immunologic stimulus to induce increased immune responses to a second antigenically different stimulus give subsequently. In this experiment, CD14.sup.+human monocytes were prepared from LeukoPaks collected by apheresis. On day 0 they were infected with either BCG-WT or BCG-disA-OE at a MOI of 5:1 for 3 hours. A third group of cells were not infected. After infection, cells were washed multiple times (every two days). After a 6-day rest period, the monocytes were re-stimulated with the TLR1/2 agonist PAM3CSK4 for 2 hours. Cells were washed repeatedly and were subsequently incubated for 24 h. Th levels of secreted IL-1 were measured in the culture supernatants by ELISA. As may be seen, while BCG-WT itself elicited statistically significantly higher levels of immune response to the second stimulus compared to uninfected cells, BCG-disA-OE elicit statistically significantly more of a response than either BCG-WT or uninfected cells.

[0095] FIG. 21 shows that BCG-disA-OE elicits a greater histone activation mark (H3K4-trimethylation) in the IL6 and TNF gene promoter regions than BCG-WT. Trained Immunity refers to the ability of a first immunologic stimulus to induce increased immune responses to a second antigenically different stimulus give subsequently. Trained immunity has been associated with epigenetic modifications, such as histone methylation, in the promoter regions of cytokines and other immune mediators. The experiment shown in FIG. 21 was performed in the same set of cells and exactly the same way as that described in FIG. 20 except that after the second stimulus with the TLR1/2 agonist PAM3CSK4 (abbreviated PAM3), cells were harvested fixed, chromatins were cross-linked and DNA was collected for chromatin immunoprecipitation analysis (ChIP) using an antibody specific for the H3K4-me3 histone methylation mark. H3K4-me3 is known to be a gene activating mark. The graph shows the relative fold change in abundance of immunoprecipitated DNA as measured by quantitative PCR using primers for the IL6 and TNF gene promoter region. As may be seen both BCG-Pasteur-disA-OE and BCG-Tice-disA-OE led to significantly greater levels of H3K4 histone trimethylation in the IL6 and TNF promoter regions than did their corresponding BCG-WT strains following challenge with the second stimulus, PAM3CSK4.

[0096] FIG. 22 shows the successful construction of BCG-Tice-disA-OE. The inventors' previous work had utilized BCG-Pasteur to construct BCG-Pasteur-disA-OE. This strain was provided to one of the inventors by Dr. Frank Collins in 1995. It is the same strain known as BCG-Pasteur-Aeras. BCG-Tice is manufactured and sold by Merck, and is the sole FDA-approved BCG available in the United States. The inventors purchase BCG-Tice, prepared electrocompetent BCG-Tice, and electroporated the pSD5-hsp60-MT3692 plasmid into BCG-Tice. The drawing shows the results of colony PCRs for 5 kanamycin-resistant candidate clones of transformed BCG-Tice and confirms the successful preparation of BCG-Tice-disA-OE by electroporation of the pSD5-hsp65-MT3692 plasmid into BCG-Tice. Note on nomenclature, the inventors had previously referred to this same plasmid pSD5-hsp60-MT3692. However, the actual promoter in this strain is the promoter for the hsp65 gene of M. leprae. Thus, the inventors now more correctly refer to the plasmid as pSD5-hsp65-MT3692.

[0097] FIG. 23 shows that clone 2 of BCG-Tice-disA-OE from the transformation experiment shown in FIG. 22 strongly expresses the disA gene. Real time PCR was used to show differential disA expression in four different BCG-Tice-disA-OE clones. Gene expression was measured in total RNA isolated from the late log phase cultures using log phase cultures using SYBR green based quantitative real-time PCR. The graphical data points represent the mean of 3 independent experiments standard error mean (SEM). M tuberculosis sigA (Rv2703) was used as an internal control. Data analysis was performed using 2.sup.CT method. Student's t test followed by Welch correction (***P<0.001; **P<0.01). The inventors created seedlots of BCG-Tice-disA-OE clone 2, and refer to this clone as simply BCG-Tice-disA-OE in all subsequent work.

[0098] FIG. 24 shows potent, statistically significantly enhanced IRF3 induction in mouse bone marrow-derived macrophages infected with BCG-Pasteur-disA-OE compared with BCG-Pasteur-WT. Mouse (C57BL/6) bone marrow-derived macrophages were infected with wild-type and disA overexpressing strains of BCG Pasteur (20 MOI) for 3 h. Cells were washed with warm DPBS to remove non-internalized bacilli and were subsequently incubated for another 3 hours. IRF3 expression was measured in total RNA isolated from the cell lysate using SYBR green based quantitative real-time PCR. The graphical data points represent the mean of 3 independent experiments standard error mean (SEM). Mouse beta-actin was used as an internal control. Data analysis was performed using 2.sup.CT method. Student's t test followed by Welch correction (***P<0.001; **P<0.01).

[0099] FIG. 25 shows that STING is required for enhanced Type I IFN (IFN-) induction in response to BCG-WT and BCG-disA-OE. Mouse (C57BL/6) bone marrow-derived macrophages from STING ablated (STING-KO) wild-type animals were infected with different strains of BCG (MOI=1:20) for 3 h. Cell were washed using warm DPBS to removed non-internalized bacilli and were subsequently incubated in for another 24 h before culture supernatants were harvested. ELISA for IFN- was performed in culture supernatants as per the manufacturer's instruction. Data points represent the mean of three independent biological experimentsstandard error mean (S.E.M.). Student's t test followed by Welch correction (**P<0.01).

[0100] FIG. 26 shows that interferon- is induced murine BMDMs, BMDCs and J774.1 macrophages in upon exposure to disA overexpressing BCG strains and that the IFN- response is statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3 h using different strains of BCG (MOI: 20). Non-internalized bacilli were washed using warm DPBS and cell were incubated for another 24 hours. IFN- levels were quantified in culture supernatants using ELISA as per manufacturer's instruction. Data points represent three independent biological experimentsstandard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001; **P<0.01; *P<0.05).

[0101] FIG. 27 shows that IL-6 is induced in mouse BMDMs, BMDCs and J774.1 macrophages in response to exposure to disA overexpressing BCG strains and that the IL-6 response is statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3 h using different strains of BCG (MOI: 20). Non-internalized bacilli were washed using warm DPBS and cell were incubated for another 24 hours. IL-6 levels were quantified in culture supernatants using ELISA as per manufacturer's instruction. Data points represent three independent biological experimentsstandard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001; **P<0.01; *P<0.05).

[0102] FIG. 28 shows that TNF is induced in mouse BMDMs, BMDCs and J774.1 macrophages in response to exposure to disA overexpressing BCG strains and that the responses are statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3 h using different strains of BCG (MOI: 20). Non-internalized bacilli were washed using warm DPBS and cell were incubated for another 24 hours. TNF levels were quantified in culture supernatants using ELISA as per manufacturer's instruction. Data points represent three independent biological experimentsstandard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001; **P<0.01; *P<0.05).

[0103] FIG. 29 shows that TNF and IFN- are induced the in the rat bladder carcinoma NBT-II cell line in response to exposure to disA overexpressing BCG strains and that the two responses are statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. NBT-II cells were infected with wild-type and recombinant strains of BCG for 3 h. Non-internalized bacilli were repeatedly washed using warm DPBS and cells were incubated for another 24 h. Culture supernatants were used for quantification of TNF and IFN-. Data points represent three independent biological experimentsstandard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.0001; **P<0.001; *P<0.05).

[0104] FIG. 30 shows that of IFN-62 , IFN-, TNF and IL-1 in are induced the in the human transitional cell papilloma RT4 bladder cancer cell line in response to exposure to disA overexpressing BCG strains and that the two responses are greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. RT4 cells were infected with wild-type and recombinant strains of BCG for 3 h. Non-internalized bacilli were repeatedly washed using warm DPBS and cells were incubated for another 24 h. Culture supernatants were used for quantification of cytokines as per manufacturer's instruction. Data points represent two independent biological experimentsstandard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001; **P<0.01; *P<0.05).

[0105] FIG. 31 shows that BCG-disA-OE stimulates increased IFN- levels in multiple bladder cancer cell lines to a greater degree than BCG-WT. The drawing shows the levels of IFN- mRNA (relative expression by the 2.sup.CT method) following exposure to BCG-WT, BCG-disA-OE, and LPS. 5637 cells are human muscle-invasive bladder cancer cells, RT4 cells are human transitional cell papilloma bladder cancer cells, and NBT-II cells are rat bladder carcinoma cells induced by N-butyl-N-(-4-hydroxybutyl) nitrosamine.

[0106] FIG. 32 shows the cytokine responses for IFN-, IFN-, IL-6, and TNF in BCG-WT and BCG-disA-OE-infected mouse lungs at different time points following aerosol infection. The drawing reveals that at most time points for most cytokines, the responses are greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. BALB/c mice were infected by the aerosol route as described in FIG. 19. Groups of mice were sacrificed at 2, 4, and 6 weeks after infection. Lung homogenates were prepared, and cytokine levels were quantified using ELISA as per manufacturer's protocol (n=4 animals/treatment groupS.E.M.). Data analysis was performed using paired t-test (***P<0.001; **P<0.01; *P<0.05).

[0107] FIG. 33 shows the cytokine responses for IFN-, IFN-, IL-6, and TNF in BCG-WT and BCG-disA-OE-infected mouse spleens at 4 weeks following aerosol infection. The drawing reveals that for most cytokines, the responses are greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains. BALB/c mice were infected by the aerosol route as described in FIG. 19. Groups of mice were sacrificed at 4 weeks after infection. Spleen homogenates were prepared, and cytokine levels were quantified using ELISA as per manufacturer's protocol (n=4 animals/treatment groupS.E.M.). Data analysis was performed using paired t-test (***P<0.001; **P<0.01; *P<0.05).

[0108] FIG. 34 shows a method to generate antibiotic-resistance gene-free recombinant BCG which overexpresses a STING-agonist. In the example given, a genetically-modified, antibiotic resistance gene-free BCG is created to overexpress the BCG disA gene and release excess c-di-AMP (a known STING agonist). However, the same strategy may be used to overexpress the Rv1354c (diguanylate cyclase that makes c-di-GMP another known STING agonist), DncV (cyclic GMP-AMP synthase from V. cholerae that makes 3-3-cGAMP another known STING agonist), cGAS (cyclic GMP-AMP synthase from humans that makes 2-3cGAMP another known STING agonist), or another similar enzyme gene that generates a STING agonist.

[0109] In Step 1 a BCG-WT strain is transformed by electroporation of the plasmid pJV53 (SEQ ID NO: 32) and selection on kanamycin-containing 7H11 agar plates. pJV53 harbors the gp60 and gp61 genes from the mycobacteriophages Che9c which encodes homologs of RecE and RecT, respectively. Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates mycobacterial homologous recombination proficiency. Step 1 yields a recombination proficient BCG as shown in the drawing. After confirmation of transformants, the positive clones will be expanded in presence of kanamycin and will be used to make electrocompetent cells. To prepare these electrocompetent cells, bacteria are grown to mid-log phase, induced with 0.2% acetamide (to upregulate the expression of the Che9c gp60 and Che9c gp61 recombineering genes) for 24 hours prior to being made electrocompetent.

[0110] In Step 2, a linearized allelic exchange substrate (AES) construct corresponding to SEQ ID NO: 33 is generated. The AES (SEQ ID NO: 33) is comprised of the dif-Hyg-dif cassette (SEQ ID NO: 34) sequence flanked by 500 bp of the 5UTR of the panCD operon on one side and 500 bp of the 3UTR of the panCD operon on the other. The AES (SEQ ID NO: 33) is constructed by cloning 500 bp of the 5UTR of the panCD operon and 500 bp of the 3UTR of the panCD operon into pUC-Hyg plasmid (SEQ ID NO: 35) yielding the plasmid pUC-Hyg-panCD-KO (SEQ ID NO: 36). pUC-Hyg-panCD-KO (SEQ ID NO: 36) is cleaved by digestion with NruI and NcoI to yield the linear AES corresponding to SEQ ID

[0111] NO: 33. Alternatively, the primers SEQ ID NO: 28 and SEQ ID NO: 29 may be used to amplify the linear AES (SEQ ID NO: 33). The linearized allelic exchange substrate (AES) construct corresponding to SEQ ID NO: 33 is then electroporated into the recombination proficient BCG, and clones are selected on hygromycin and kanamycin-containing 7H11 agar plates. This step yields BCG harboring the panCD KO cassette in which the AES (SEQ ID NO: 33) has integrated into the panCD operon of the chromosome by homologous recombination.

[0112] In Step 3 several hundred colonies of BCG harboring the panCD KO cassette are replica plated on both (i) kanamycin-containing 7H11 agar plates and (ii) kanamycin-containing 7H11 agar supplemented with pantothenate (24 g/ml). Kan-resistant, pantothenate auxotrophic clones are selected that only grow on kanamycin-containing 7H11 agar supplemented with pantothenate (24 g/ml) and fail to grow on kanamycin-containing 7H11 agar plates lacking pantothenate. During this step the natural action of the mycobacterial Xer recombinase which recognizes and recombines at dif sites leads to excision and loss of the hygromycin cassette yielding clones which are Pantothenate auxotrophs harboring pJV53 as shown.

[0113] In Step 4, pantothenate auxotrophs harboring pJV53 are plated on 7H11 agar plates containing sucrose to select for loss of pJV53 which harbors the sacB gene (conferring lethality in the presence of sucrose). Sucrose-resistant clones are selected, and these are confirmed to be kanamycin-susceptible. This yield clones which are Pantothenate auxotrophs free of pJV53.

[0114] In Step 5, electrocompetent Pantothenate auxotrophs free of pJV53 are prepared. The plasmid pSD5.phsp65-disA.panCD-No Kan (SEQ ID NO: 31) is generated as described in FIG. 36. SEQ ID NO: 31 is electroporated into Pantothenate auxotrophs free of pJV53, and clones are plated on 7H11 agar free of pantothenate to yield the desired Pantothenate auxotroph harboring a disA-OE plasmid. Candidate clones are confirmed by PCR of relevant genes and by whole genome sequencing.

[0115] FIG. 35 shows the molecular structure of the DNA fragment containing the panCD allelic exchange substrate (AES) which is SEQ ID NO: 33.

[0116] FIG. 36 shows the strategy used to generate pSD5.hsp65-disA.panCD-No Kan (SEQ ID NO: 31). The scheme replaces Kan cassette pSD5.hsp65-disA.Kan (SEQ IN NO: 30) with the panCD operon to generate pSD5.hsp65-disA.panCD-No Kan (SEQ ID NO: 31).

[0117] FIG. 37 shows the molecular structure of the pJV53, the recombineering plasmid which is SEQ ID NO: 32

[0118] FIG. 38 shows the molecular structure of the pUC-Hyg, a plasmid with dif sites flanking a Hyg cassette which is SEQ ID NO: 35. pUC-Hyg is used to generate the plasmid pUC-Hyg-panCD-KO (SEQ ID NO: 36).

[0119] FIG. 39 shows the molecular structure of the plasmid pUC-Hyg-panCD-KO which is SEQ ID NO: 36. pUC-Hyg-panCD-KO is generated by cloning 500 bp of the panCD 5UTR on one flank of the Hyg cassette, and cloning 500 bp of the panCD 3UTR the other flank.

[0120] FIG. 40 shows the molecular structure of the plasmid pSD5.hsp65-disA.Kan which is SEQ ID NO: 30.

[0121] FIG. 41 shows the molecular structure of the plasmid pSD5.hsp65-disA.panCD-No Kan which is SEQ ID NO: 31. This plasmid is generated using the scheme illustrated in FIG. 36.

[0122] FIG. 42 shows some of the nucleic acid and protein sequences used in the present invention.

[0123] FIG. 43 shows a description of the nucleic acid and protein sequences used in the present invention.

[0124] FIG. 44 shows the number of positive specimens.

DETAILED DESCRIPTION OF THE INVENTION

[0125] In some embodiments, the present invention relates to genetic alterations of Mycobacterium bovis BCG (hereafter, BCG) which generate recombinant BCG (hereafter rBCG) strains. These strains have greater potency as (i) tuberculosis vaccines and/or (ii) immunotherapies for non-muscle invasive bladder cancer (NMIBC). Some embodiments of the present invention relate to BCG strains that synthesize and secrete high levels of cyclic dinucleotides (CDNs) which are known to elicit valuable immunomodulatory responses from human phagocytic cells such as macrophages, dendritic cells, and others. Another embodiment of this invention is to combine genetic modifications of BCG to generate multivalent CDN-overexpression modifications that include addition of novel CDN-synthesizing genetic material and/or mutations of endogenous BCG phosphodiesterase genes or genetic domains that will enhance the accumulation and release of CDNs.

[0126] BCG

[0127] BCG (bacillus Calmette Guerin) is a mutant version of Mycobacterium bovis generated by the French microbiologists Calmette and Guerin in 1921 by 13 years of serial passage of virulent M bovis. Between 1921 and 1960 BCG was carried by serial passage in numerous world laboratories, until defined seedlots were established and banked in reference laboratories. As such, many dozen variants of BCG exist worldwide such as BCG Pasteur, BCG Tice, BCG Tokyo, BCG Danish, BCG Montreal, etc. The majority of existing BCG strains have now been defined by whole genome sequencing. Major differences between virulent M. bovis and the various BCG strains include the deletion of at least 15 regions of difference that comprise genomic deletions in BCG compared with virulent M tuberculosis. Key regions of difference in the development of BCG were RD1 (9.5 kb deletion leading to loss of the Esx-1 secretion system and inability to release antigens ESAT-6 and CFP-10) and RD3 (9.2 kb deletion). Regions of difference RD4-RD11 are absent in all BCG strains compared with virulent M tuberculosis.

[0128] Since the 1920s, BCG has been used as a vaccine for prevention of tuberculosis (TB). In 2004 it was estimated that BCG was given to about 100 million children, hence since its introduction BCG has been given to approximately 5 billion humans and as such is the most widely utilized vaccine in history. It is most commonly given intradermally at birth, and to date it is still given in most countries except the United States, Canada, and parts of Europe. BCG has been shown to reduce the incidence of childhood disseminated TB, but BCG-vaccinated individuals are not fully protected from the risk of TB.

[0129] Since 1977, BCG has also achieved wide use as a cancer immunotherapy for non-muscle invasive bladder cancer (NMIBC). It is given intravesically weekly for six weeks and in some instances such as high-risk disease it is given as maintenance therapy weekly for three weeks at 3, 6, 12, 18, 24, 30, and 36 months after initial therapy. Intravesical BCG has been shown to (i) induce a mononuclear cell infiltrate comprised predominantly of CD4 T cells and macrophages, (ii) increase the expression of interferon gamma (IFN) in the bladder, and (iii) increase urinary cytokine levels of IL-1, IL-2, IL-6, IL-8, IL-12, IFN, and TNF.

[0130] Despite the wide global use of BCG as (i) a vaccine for TB and (ii) an immunotherapy for NMIBC, there is considerable room for improvement in its efficacy. For TB, BCG gives only partial protection predominantly against childhood disseminated tuberculosis. For NMIBC, approximately 30% of patients have BCG-resistant disease. These individuals require riskier treatments with systemic chemotherapy and have higher rates of progression to more invasive forms of bladder cancer.

[0131] Urothelial Cancer

[0132] Urothelial cancer of the bladder is the most common malignancy of the urinary tract. It is the fourth most common cancer in males and 11th most common in females. It is estimated that approximately 79,000 new cases of bladder cancer will be diagnosed in the USA in 2017, associated with 19,870 deaths. Although the estimated five-year survival for bladder cancer patients is 78%, the rates decline dramatically for patients with locally advanced or metastatic disease. Approximately 75% of patients with bladder cancer present with a disease that is confined to the mucosa (stage Ta, carcinoma in situ) or submucosa (stage T1), know) as non-muscle invasive bladder cancer (NMIBC). Transurethral resection is the initial treatment of choice for NMIBC. For patients with muscle invasive bladder cancer (MIBC; T2 or greater), the first-line treatment option is platinum-containing chemotherapy followed by bladder removal. For those patients with NMIBC who do not respond to intravesical treatments, there is high risk of progression to MIBC. Thus, the high rates of recurrence and significant risk of progression mandate that additional therapy be implemented. Improving clinical outcomes for patients with high risk-NMIBC therefore requires the development of novel treatments.

[0133] Intra-vesical administration of Bacillus-Calmette Guerin (BCG), developed in the 1970s for NMIBC, provided the first successful immunotherapy against an established solid cancer, and it remains the standard of care for patients with NMIBC. (Askeland E J, Newton M R, O'Donnell M A, Luo Y. Bladder Cancer Immunotherapy: BCG and Beyond. Adv Urol. 2012; 2012:181987. PMID: 22778725. Morales A. BCG: A throwback from the stone age of vaccines opened the path for bladder cancer immunotherapy. Can J Urol. 2017; 24:8788-8793. PMID: 28646932). The exact mechanism of the anti-tumor effects of BCG, which is an attenuated strain of Mycobacterium bovis, remain unclear, but it is believed to orchestrate a vigorous immune cellular and humoral immune response, predominantly Th1 response, after binding to the urothelium through fibronectin and integrin a5131 (Redelman-Sidi G, Glickman M S, Bochner B H. The mechanism of action of BCG therapy for bladder cancera current perspective. Nat Rev Urol. 2014; 11:153-62. PMID: 24492433). However, typical complete response rates for BCG treatment are 55-65% for papillary tumors and 70-75% for carcinoma in situ (CIS). (Askeland E J, Newton M R, O'Donnell M A, Luo Y. Bladder Cancer Immunotherapy: BCG and Beyond. Adv Urol. 2012;2012:181987. PMID: 22778725. Morales A. BCG: A throwback from the stone age of vaccines opened the path for bladder cancer immunotherapy. Can J Urol. 2017; 24:8788-8793. PMID: 28646932). The burden of patients with BCG unresponsive and relapsing disease and of those intolerant of treatment has therefore prompted the need for further improving the efficacy of BCG against NMIBC.

[0134] CDNs are Important PAMPs and DAMPs that Generate Valuable Immune Responses for TB and NMIBC.

[0135] Bacterial pathogen-associated molecular patterns (PAMPs). Human cells utilize an innate immune monitoring system known as the cytosolic surveillance program (CSP) to detect nucleic acid including cyclic dinucleotides in the cytosol. Originally characterized as a viral defense system, the CSP has now been shown to be important in anti-bacterial defenses particularly against intracellular bacteria such as Mycobacterium tuberculosis, Listeria monocytogenes, Salmonella species, and others. Cytosolic pattern recognition receptors (PRRs) including STING, cGAS, DDX41 and many others are capable of binding to cytosolic CDNs and nucleic acids leading to their activation. A key signaling event is STING activation which leads to activation of TBK1 and IRF3 and subsequent upregulation of type I interferon expression. STING activation by cyclic dinucleotides also leads to the induction of STAT6 which induces chemokines such as CCL2 and CCL20 independently of the TBK1-IRF3 pathway. STING activation is also believed to activate the transcription factor NFB through the B kinase (IKK) activation.

[0136] Human danger associated molecular patterns (DAMPs). Cyclic cGAMP (cGAS) synthase is a cytosolic PRR which recognized cytosolic DNA. Upon binding to DNA it undergoes a conformational change that activates its core enzymatic activity which is to catalyze the formation of 23 cGAMP. 23 cGAMP in turn is a potent DAMP which activates the STING-TBK1-IRF3 axis leading to increased type 1 interferon expression as well as the STAT6 activation and IKK activation.

[0137] STING-mediated mechanism of CDN-triggered immune responses. Type I IFNs, produced both by innate immune cells in the tumor microenvironment and by the tumor cells themselves, are known to mediate anti-tumor effects against several malignancies, due to their ability to intervene in all phases of cancer immune-editing. (Zitvogel L, Galluzzi L, Kepp O, Smyth M J, Kroemer G. Type I interferons in anticancer immunity. Nat Rev Immunol. 2015;15:405-14. PMID: 26027717). STING (stimulator of interferon genes), is a major regulator of Type I IFN innate immune responses to pathogens, following recognition of cytosolic DNA by the sensor cyclic GMP-AMP synthase (cGAS). cGAS catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which in turn functions as a second messenger that binds and activates STING. (Zhao G N, Jiang D S, Li H. Interferon regulatory factors: at the crossroads of immunity, metabolism, and disease. Biochim Biophys Acta. 2015;1852:365-78. PMID: 24807060). Novel anticancer immunotherapies based on recombinant type I IFNs, type I IFN-encoding vectors, type I IFN-expressing cells, and STING agonists are therefore currently being developed as novel tumor immunotherapies.

[0138] Overexpression of the PAMP immunomodulator, 3-5 c-di-AMP. 3-5 c-di-AMP is a strong inducer of the STING-TBK1-IRF3 axis. It is produced by mycobacteria including BCG by the disA gene which encodes the DisA protein (BCG protein WP 010950916.1 in BCG, M tuberculosis protein Rv3586 or P9WNW5.1). Mycobacterium tuberculosis (M.tb) synthesizes and secretes c-di-AMP, which activates the interferon regulatory factor (IRF) pathway and Type I IFN responses through STING-signaling and cGAS. (Ahmed D, Cassol E. Role of cellular metabolism in regulating type I interferon responses: Implications for tumour immunology and treatment. Cancer Lett. 2017; 409:20-29. PMID: 28888999.). c-di-AMP overexpressing M. tb strains showed attenuation of TB in a mouse model. As a mucosal adjuvant, c-di-AMP exerts immune stimulatory effects causing maturation of dendritic cells, up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines, and strong Th1, Th17 and CD8 T cell responses against pathogens. A c-di-AMP-overexpressing BCG strain (rBCG-disA or BCG-disA-OE) has been constructed and surprisingly found that it produced a significantly higher IRF and IFN- response than BCG itself, indicating that bacteria-derived c-di-AMP gains access to the host cell cytosol despite the absence of the ESX-1 protein secretion system. (Ahmed D, Cassol E. Role of cellular metabolism in regulating type I interferon responses: Implications for tumour immunology and treatment. Cancer Lett. 2017; 409:20-29. PMID: 28888999.). These findings suggest that rBCG strains modified to overexpress c-di-AMP could induce better protective immunity against bladder tumors than BCG itself.

[0139] Induction of pro-inflammatory Th1 cytokines in mouse bone marrow-derived macrophages (BMDMs) in response to BCG overexpressing M.tb disA (MT3692): M.tb genome encodes a di-adenylate cyclase enzyme (DisA, also called DacA, P9WNW5.1 in the UniProtKB/Swiss-Prot databases) that synthesizes c-di-AMP from ATP or ADP. The BCG protein WP_010950916.1 (NCBI reference number) is 100% identical to M. tuberculosis DisA. M. tb strains overexpressing disA intoxicate macrophages by releasing excessive c-di-AMP, a unique bacterial PAMP that activates STING-dependent IFN-production. (Ahmed D, Cassol E. Role of cellular metabolism in regulating type I interferon responses: Implications for tumour immunology and treatment. Cancer Lett. 2017; 409:20-29. PMID: 28888999.). To expand the antigenic repertoire of a non-pathogenic vaccine strain, BCG Pasteur was transformed with a kanamycin-resistance (Kan-R)-conferring plasmid that harbors the disA gene (M tuberculosis Rv3586 or MT3692) from M. tb (the M. tb and BCG disA genes are 100% identical) fused to the strong mycobacterial promoter, Phs.sub.p60. Addition of this plasmid to BCG-Pasteur increased the level of disA mRNA by 50-fold (FIG. 1b). The closely related M.tb-disA-OE strain releases 15-fold more c-di-AMP into the macrophage cytosol than wild type M. tb. (FIG. 1a), and hence it is expected that BCG-disA-OE also releases significantly more c-di-AMP into the host cytosol. These disA overexpressor recombinants (rBCG or BCG-disA-OE) were better inducers of STING-dependent IFN- as compared to the parental strain. Most importantly as reported in PCT/US2016/017248, filed Feb. 10, 2016, guinea pigs vaccinated with rBCG were significantly better protected against aerosol infection with virulent M.tb, suggesting improved protective efficacy over existing BCG strain.

[0140] As shown in FIG. 2, immune responses elicited by BCG-Pasteur disA-OE were tested in an in vitro macrophage infection model. BMDMs from C57BL/6 mice infected with BCG-Pasteur disA-OE showed significant upregulation of IFN-, TNF-, IL-6 and IL-2 in comparison to uninfected or wild-type BCG infected macrophages.

[0141] As shown in FIG. 3, augmented c-di-AMP-based STING activation was confirmed in RAWBlue ISG macrophages. RAWBlue macrophages showed increased IRF3 levels when infected with BCG-Pasteur disA-OE, as compared to parental control.

[0142] As shown in FIG. 4, a significant increase in secreted pro-inflammatory cytokines (TNF-, IL-6 and IL-1) was found in culture supernatants of BCG-Pasteur-disA-OE infected mouse BMDMs. These findings indicate that BCG-Pasteur-disA-OE with increased antigenic repertoire acts like a STING agonist, and hence a potent inducer of STING-dependent Type I IFNs. Furthermore, the immune responses in macrophages in response to BCG-Pasteur disA-OE were skewed towards Th1, a phenotype largely attributed for control of NMIBC by BCG immunotherapy.

[0143] As shown in FIG. 5, BCG-disA-OE elicits anti-tumor immune responses in human bladder carcinoma (RT4) cells. BCG-Pasteur-disA-OE was tested to determine whether it elicits similar immune responses in bladder cancer (BC) cells, in comparison to WT strains BCG-Pasteur and OncoTICE (the current immunotherapeutic BCG strain). Human RT4 BC cells, derived from human NMIBC tumors, were challenged with the wild-type (both Pasteur and TICE) and recombinant BCG Pasteur disA-OE strain at 1:20 (RT4::BCG) for 3 h, and differential gene expression profile was determined in comparison to uninfected cells. Key immune mediators such as, monocyte chemoattractant protein 1 (MCP-1)/CCL2, IFN- and IL-1 were found to be significantly increased in bladder cancer cells exposed to BCG-Pasteur-disA-OE compared to responses to wild type strains.

[0144] As shown in FIG. 6, an experimental system was set up to test whether intravesical BCG-disA-OE immunotherapy leads to heightened Th1 responses and anti-tumor efficacy in the MNU carcinogen model of NMIBC. Results from the aforementioned experiments with RT4 cells encouraged the inventors to test the relative therapeutic efficacy of BCG-Pasteur disA OE in an in vivo rat NMIBC model, pioneered in Bivalacqua lab. (Kates M, Nirschl T, Sopko N A, Matsui H, Kochel C M, Reis L O, Netto G J, Hoque M O, Hahn N M, McConkey D J, Baras A S, Drake C G, Bivalacqua T J. Intravesical BCG Induces CD4(+) T-Cell Expansion in an Immune Competent Model of Bladder Cancer. Cancer Immunol Res. 2017; 5:594-603. PMID: 28588015). In this model, N-methyl-N-nitrosourea (MNU), a carcinogenic alkylating agent, is used to induce urothelial cancer in female Fischer rats.

[0145] As can be seen in FIG. 7, BCG-disA-OE has significant immunotherapeutic effects in the rat bladder cancer model. Urothelial dysplasia develops within eight weeks of MNU instillation, and by the 16th week after the first instillation, all rats display carcinoma-in-situ, papillary Ta, or high-grade T1 urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human urothelial cancer. Using this model, the Bivalacqua lab showed that intravesical BCG immunotherapy lead to a large, transient rise in the CD4.sup.+ T cell population in the urothelium. (Kates M, Nirschl T, Sopko N A, Matsui H, Kochel C M, Reis L O, Netto G J, Hoque M O, Hahn N M, McConkey D J, Baras A S, Drake C G, Bivalacqua T J. Intravesical BCG Induces CD4(+) T-Cell Expansion in an Immune Competent Model of Bladder Cancer. Cancer Immunol Res. 2017;5:594-603. PMID: 28588015). Intravesical instillation of BCG-disA-OE strain was performed in MNU-treated rats, administered sequentially every week for 6 weeks starting eight weeks after MNU induction when tumors are visible. Bladder tumors were staged by a GU pathologist according to WHO-ISUP classifications with percent tumor involvement (sum of Ta, T1 and CIS) calculated for each group according to criteria as described. (Kates M, Nirschl T, Sopko N A, Matsui H, Kochel C M, Reis L O, Netto G J, Hoque M O, Hahn N M, McConkey D J, Baras A S, Drake C G, Bivalacqua T J. Intravesical BCG Induces CD4(+) T-Cell Expansion in an Immune Competent Model of Bladder Cancer. Cancer Immunol Res. 2017;5:594-603. PMID: 28588015). A significant decrease in tumor involvement index in rats treated with BCG-Pasteur disA-OE was found in comparison to bladders from untreated or BCG-Pasteur treated rats.

[0146] As can be seen in FIG. 8, BCG-disA-OE induces a characteristic cytokine and chemokine signature in rat bladders undergoing immunotherapy. Rat urinary bladders from rats treated with BCG-disA-OE showed a significant induction of IFN-/, IFN-, IL-1, TNF-, TGF-, iNOS, IP-10, MCP-1 and MIP-la in comparison to untreated or BCG-Pasteur treated rats.

[0147] As shown in FIG. 9, evidence was found for increased infiltration of CCL2.sup.+ macrophages, Nos2.sup.+ and IL-1.sup.+ M1 macrophages, accompanied by increased IL-6 and IFN-expression in bladders of rats treated with BCG-Pasteur-disA-OE. Interestingly, increased levels of IP-10 were found, which together with increased IFN- is known to promote a strong T cell recruitment at the site of infection and inflammation.

[0148] FIG. 10 shows a summary of the cytokine expression level changes observed with BCG-disA-OE versus BCG-WT in primary cells, cancer cell lines, and in rat bladder cancer tissues. As can be seen, cytokines associated with Th1 T cell and M1 macrophage expansion, two Type 1 interferons, and three pro-inflammatory chemokines were significantly unregulated by BCG-disA-OE compared to BCG-WT (2-fold to 30-fold) across these cells, cell lines and tissues. In contrast, cytokines associated with Th2 T cell and M2 macrophage expansion were generally down-regulated by BCG-disA-OE in comparison to BCG-WT (1-fold to 10-fold).

[0149] As shown in FIG. 11, BCG immunotherapy may be effective via three immune mechanisms: (i) increased generation of tumor-specific cytotoxic CD8 T cells, (ii) cytokine environment which promotes macrophage-mediated CD4 cell activation against tumor antigens, and (iii) macrophage M1 shift promoting enhanced tumoricidal activity. The findings reported herein strongly indicate that BCG overexpressing c-di-AMP is taken up by bladder tumor cells, and myeloid cells that are either resident or recruited to the tumor microenvironment, and induces host immune responses, including activation of STING and Type I IFN responses, and NF-KB signaling, that promotes secretion of cytokines and chemokines, macrophage recruitment and apoptotic mechanisms, all of which collectively reduce tumor progression.

[0150] As shown in FIG. 12, in addition to overexpression of disA generating increased levels of the PAMP molecule c-di-AMP, there are additional recombinant DNA modification which may be made to BCG to enhance its production of other PAMP and DAMP molecules. As shown in the FIG. 12, genes for other CDN cyclases(i) the GGDEF domain of the BCG_RS07340 protein orM tuberculosis Rv1354c protein (100% identical to each other), (ii) the Vibrio cholerae DncV protein, Q9KVG7 in Swiss-Prot, which is a 2-5c-GAMP synthase, and (iii) the human cGAS protein Q8N884 in Swiss-Prot which is a 2-3 cGAMP synthasemay be added to BCG. These added CDN cyclase genes may be added alone or in combination. Such combinations would represent multivalent CDN overexpressing BCG. Also, as shown in FIG. 13, BCG possess several CDN phosphodiesterase genes or genes which contain phosphodiesterase domains. Recombinant technology methods to remove these endogenous phosphodiesterase genes and intragenic phosphodiesterase domains: (i) the BCG WP_003414507 gene which encodes a CDN PDE in BCG that is 100% identical to the M. tuberculosis Rv2837c (also called CdnP or CnpB), (ii) the DNA encoding the EAL domain of protein BCG_RS07340 (previously BCG_1416c) which is 100% identical to the known CDN PDE M. tuberculosis Rv1354c protein, and (iii) the gene encoding BCG AHM07112 which is homologous the known CDN PDE M. tuberculosis Rv1357c. Removal of the genes encoding these PDEs will serve to further increase the levels of CDN PAMP and DAMP molecules produced by the rBCG strains disclosed herein.

[0151] SEQ ID NO: 1

[0152] Diadenylate cyclase DisA from BCG and other related mycobacteria, amino acid sequence (358 amino acids; BCG protein A0Q92 RS18745; NCBI Reference Sequence: NZ_CUWL01000001.1). The identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as protein Rv3586 or MT3692, and in Mycobacterium bovis as protein Mb3617.

TABLE-US-00001 MHAVTRPTLREAVARLAPGTGLRDGLERILRGRTGALIVLGHDENVEAIC DGGFSLDVRYAATRLRELCKMDGAVVLSTDGSRIVRANVQLVPDPSIPTD ESGTRHRSAERAAIQTGYPVISVSHSMNIVTVYVRGERHVLTDSATILSR ANQAIATLERYKTRLDEVSRQLSRAEIEDFVTLRDVMTVVQRLELVRRIG LVIDYDVVELGTDGRQLRLQLDELLGGNDTARELIVRDYHANPEPPSTGQ INATLDELDALSDGDLLDFTALAKVFGYPTTTEAQDSTLSPRGYRAMAGI PRLQFAHADLLVRAFGTLQGLLAASAGDLQSVDGIGAMWARHVREGLSQL AESTISDQ

[0153] SEQ ID NO: 2

[0154] Diadenylate cyclase disA from BCG and other related mycobacteria, DNA sequence (1077 nucleotides [358 codons, 1 stop codon]; encodes BCG gene A0Q92_RS18745; NCBI Reference Sequence: NZ_CUWL01000001.1) Identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as gene Rv3586 or MT3692, Mycobacterium bovis as gene Mb3617.

TABLE-US-00002 1 atgcacgctgtgactcgtccgaccctgcgtgaggctgtcgcccgcctagccccgggcact 61 gggctgcgggacggcctggagcgtatcctgcgcggccgcactggtgccctgatcgtgctg 121 ggccatgacgagaatgtcgaggccatctgcgatggtggcttctccctcgatgtccgctat 181 gcagcaacccggctacgcgagctgtgcaagatggacggcgccgtggtgctgtccaccgac 241 ggcagccgcatcgtgcgggccaacgtgcaactggtaccggatccgtcgatccccaccgac 301 gaatcggggacccggcaccgctcggccgagcgggccgcgatccagaccggttacccggtg 361 atctcagtgagccactcgatgaacatcgtgaccgtctacgtccgcggggaacgtcacgta 421 ttgaccgactcggcaaccatcctgtcgcgggccaaccaggccatcgcaaccctggagcgg 481 tacaaaaccaggctcgacgaggtcagccggcaactgtccagggcagaaatcgaggacttc 541 gtcacgctgcgcgatgtgatgacggtggtgcaacgcctcgagctggtccggcgaatcggg 601 ctggtgatcgactacgacgtggtcgaactcggcactgatggtcgtcagctgcggctgcag 661 ctcgacgagttgctcggcggcaacgacaccgcccgggaattgatcgtgcgcgattaccac 721 gccaacccggaaccaccgtccacggggcaaatcaatgccaccctggacgaactggacgcc 781 ctgtcggacggcgacctcctcgatttcaccgcgctggcaaaggttttcggatatccgacg 841 accacggaagcgcaggattcggcgctgagcccgcgtggctaccgcgcgatggccggtatc 901 ccccggctccagttcgcccatgccgacctgctggtccgggcgttcggaacgttgcagggt 961 ctgctggcggccagcgccggcgatctgcaatcagtggacggcatcggcgccatgtgggcc 1021 cgtcatgtgcgcgatgggttgtcacagctggcggaatcgaccatcagcgatcaataa

[0155] SEQ ID NO: 3.

[0156] Plasmid pSD5B-P.sub.hsp60::disA which is an episomally replicating E. coli-mycobacterial shuttle plasmid that overexpresses the BCG disA gene from the P.sub.hsp60 promoter, DNA sequence. (7742 nucleotides; promoter P.sub.hsp60 DNA comprised of a portion of the M. leprae hsp65 gene nucleotides 13 to 181) is underlined; disA coding sequence nucleotides 242 to 1318; ATG start codon and TAA stop codon shown in boldface, underline).

TABLE-US-00003 GGATCCTTCTAGAATTCCGGAATTGCACTCGCCTTAGGGGAGTGCTAAAAATGATCCTGGCACTCGCGATCAGCGAG 1-77 TGCCAGGTCGGGACGGTGAGACCCAGCCAGCAAGCTGTGGTCGTCCGTCGCGGGCACTGCACCCGGCCAGCGTAAGT 78-154 AATGGGGGTTGTCGGCACCCGGTGACCTAGACACATGCATGCATGCTTAATTAATTAAGCGATATCCGGAGGAATCA 155-231 CTTCCATATGATGCACGCTGTGACTCGTCCGACCCTGCGTGAGGCTGTCGCCCGCCTAGCCCCGGGCACTGGGCTGC 232-308 GGGACGGCCTGGAGCGTATCCTGCGCGGCCGCACTGGTGCCCTGATCGTGCTGGGCCATGACGAGAATGTCGAGGCC 309-385 ATCTGCGATGGTGGCTTCTCCCTCGATGTCCGCTATGCAGCAACCCGGCTACGCGAGCTGTGCAAGATGGACGGCGC 386-462 CGTGGTGCTGTCCACCGACGGCAGCCGCATCGTGCGGGCCAACGTGCAACTGGTACCGGATCCGTCGATCCCCACCG 463-539 ACGAATCGGGGACCCGGCACCGCTCGGCCGAGCGGGCCGCGATCCAGACCGGTTACCCGGTGATCTCAGTGAGCCAC 540-616 TCGATGAACATCGTGACCGTCTACGTCCGCGGGGAACGTCACGTATTGACCGACTCGGCAACCATCCTGTCGCGGGC 617-693 CAACCAGGCCATCGCAACCCTGGAGCGGTACAAAACCAGGCTCGACGAGGTCAGCCGGCAACTGTCCAGGGCAGAAA 694-770 TCGAGGACTTCGTCACGCTGCGCGATGTGATGACGGTGGTGCAACGCCTCGAGCTGGTCCGGCGAATCGGGCTGGTG 771-847 ATCGACTACGACGTGGTCGAACTCGGCACTGATGGTCGTCAGCTGCGGCTGCAGCTCGACGAGTTGCTCGGCGGCAA 848-924 CGACACCGCCCGGGAATTGATCGTGCGCGATTACCACGCCAACCCGGAACCACCGTCCACGGGGCAAATCAATGCCA 925-1001 CCCTGGACGAACTGGACGCCCTGTCGGACGGCGACCTCCTCGATTTCACCGCGCTGGCAAAGGTTTTCGGATATCCG 1002-1078 ACGACCACGGAAGCGCAGGATTCGACGCTGAGCCCGCGTGGCTACCGCGCGATGGCCGGTATCCCCCGGCTCCAGTT 1079-1155 CGCCCATGCCGACCTGCTGGTCCGGGCGTTCGGAACGTTGCAGGGTCTGCTGGCGGCCAGCGCCGGCGATCTGCAAT 1156-1232 CAGTGGACGGCATCGGCGCCATGTGGGCCCGTCATGTGCGCGAGGGGTTGTCACAGCTGGCGGAATCGACCATCAGC 1233-1309 GATCAATAAACGCGTTCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT 1310-1386 GGCGAATGGCGCTTTGCCTGGTTTCCGGTCGAAGCTTGGCCGGATCTAAAGTTTTGTCGTCTTTCCAGACGTTAGTA 1387-1463 AATGAATTTTCTGTATGAGGTTTTGCTAAACAACTTTCAACAGTTTCAGCGGAGTGAGAATAGAAAGGAACAACTAA 1464-1540 AGGAATTGCGAATAATAATTTTTTCACGTTGAAAATCTCCAAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGTATC 1541-1617 GGTTTATCAGCTTGCTTTCGAGGTGAATTTCTTAAACAGCTTGATACCGATAGTTGCGCCGACAATGACAACAACCA 1618-1694 TCGCCCACGCATAACCGATATATTCGGTCGCTGAGGCTTGCAGGGAGTCAAAGGCCGCTTTTGCGGGGATCCGCTCG 1695-1771 GAGGCGCGGTCGCGGCGCGGCTGTGGCATGTCGGGGCGTGCCGCTCCCCCGGCGCCGCCCATCGGCCCGCCCATTGG 1772-1848 CATTCCGCCCATGCCGCCCATCATTCCTGTGGAGCCAGAACTGATCCAGCCTGTGCCACAGCCGACAGGATGGTGAC 1849-1925 CACCATTTGCCCCATATCACCGTCGGTACTGATCCCGTCGTCAATAAACCGAACCGCTACACCCTGAGCATCAAACT 1926-2002 CTTTTATCAGTTGGATCATGTCGGCGGTGTCGCGGCCAAGACGGTCGAGCTTCTTCACCAGAATGACATCACCTTCC 2003-2079 TCCACCTTCATCCTCAGCAAATCCAGCCCTTCCCGATCTGTTGAACTGCCGGATGCCTTGTCGGTAAAGATGCGGTT 2080-2156 AGCTTTTACCCCTGCATCTTTGAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAAT 2157-2233 CGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTT 2234-2310 TGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTT 2311-2387 CGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCT 2388-2464 GATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAA 2465-2541 AAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA 2542-2618 TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCA 2619-2695 TGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATT 2696-2772 ACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGC 2773-2849 GATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATA 2850-2926 TTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGC 2927-3003 ATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCT 3004-3080 CATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT 3081-3157 CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGA 3158-3234 ATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAG 3235-3311 CAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGG 3312-3388 CTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGT 3389-3465 GGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCT 3466-3542 GGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTAGCGG 3543-3619 AGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTG 3620-3696 CACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTT 3697-3773 CGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGA 3774-3850 AGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAA 3851-3927 TCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTG 2928-4004 TTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTC 4005-4081 CGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTA 4082-4158 ACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGA 4159-4235 GTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGT 4236-4312 TACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAG 4313-4389 CAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAA 4390-4466 CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAAGTGCTCATCATTG 4467-4543 GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA 4544-4620 CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAA 4621-4697 AAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG 4698-4774 GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC 4775-4851 CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCC 4852-4928 CTTTCGTCTTCAAGAATTCCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTG 4929-5005 TTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCG 5006-5082 GAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGGAATTCCCATCGAGCCGAGAACGTTATCGAAGTTGGTCA 5083-5159 TGTGTAATCCCCTCGTTTGAACTTTGGATTAAGCGTAGATACACCCTTGGACAAGCCAGTTGGATTCGGAGACAAGC 5160-5236 AAATTCAGCCTTAAAAAGGGCGAGGCCCTGCGGTGGTGGAACACCGCAGGGCCTCTAACCGCTCGACGCGCTGCACC 5237-5313 AACCAGCCCGCGAACGGCTGGCAGCCAGCGTAAGGCGCGGCTCATCGGGCGGCGTTCGCCACGATGTCCTGCACTTC 5314-5390 GAGCCAAGCCTCGAACACCTGCTGGTGTGCACGACTCACCCGGTTGTTGACACCGCGCGCGGCCGTGCGGGCTCGGT 5391-5467 GGGGCGGCTCTGTCGCCCTTGCCAGCGTGAGTAGCGCGTACCTCACCTCGCCCAACAGGTCGCACACAGCCGATTCG 5468-5544 TACGCCATAAAGCCAGGTGAGCCCACCAGCTCCGTAAGTTCGGGCGCTGTGTGGCTCGTACCCGCGCATTCAGGCGG 5545-5621 CAGGGGGTCTAACGGGTCTAAGGCGGCGTGTACGCGGCCACAGCGGCTCTCAGCGGCCCGGAAACGTCCTCGAAACG 5622-5698 ACGCATGTGTTCCTCCTGGTTGGTACAGGTGGTTGGGGGTGCTCGGCTGTCGCGGTTGTTCCACCACCAGGGCTCGA 5699-5775 CGGGAGAGCGGGGGAGTGTGCAGTTGTGGGGTGGCCCCTCAGCGAAATATCTGACTTGGAGCTCGTGTCGGACCATA 5776-5852 CACCGGTGATTAATCGTGGTCTACTACCAAGCGTGAGCCACGTCGCCGACGAATTTGAGCAGCTCTGGCTGCCGTAC 5853-5929 TGGCCGCTGGCAAGCGACGATCTGCTCGAGGGGATCTACCGCCAAAGCCGCGCGTCGGCCCTAGGCCGCCGGTACAT 5930-6006 CGAGGCGAACCCAACAGCGCTGGCAAACCTGCTGGTCGTGGACGTAGACCATCCAGACGCAGCGCTCCGAGCGCTCA 6007-6083 GCGCCCGGGGGTCCCATCCGCTGCCCAACGCGATCGTGGGCAATCGCGCCAACGGCCACGCACACGCAGTGTGGGCA 6084-6160 CTCAACGCCCCTGTTCCACGCACCGAATACGCGCGGCGTAAGCCGCTCGCATACATGGCGGCGTGCGCCGAAGGCCT 6161-6237 TCGGCGGCCGTCGACGGCGACCGCAGTTACTCAGGCCTCATGACCAAAAACCCCGGCCACATCGCCTGGGAAACGGA 6238-6314 ATGGCTCCACTCAGATCTCTACACACTCAGCCACATCGAGGCCGAGCTCGGCGCGAACATGCCACCGCCGCGCTGGC 6315-6391 GTCAGCAGACCACGTACAAAGCGGCTCCGACGCCGCTAGGGCGGAATTGCGCACTGTTCGATTCCGTCAGGTTGTGG 6392-6468 GCCTATCGTCCCGCCCTCATGCGGATCTACCTGCCGACCCGGAACGTGGACGGACTCGGCCGCGCGATCTATGCCGA 6469-6545 GTGCCACGCGCGAAACGCCGAATTCCCGTGCAACGACGTGTGTCCCGGACCGCTACCGGACAGCGAGGTCCGCGCCA 6546-6622 TCGCCAACAGCATTTGGCGTTGGATCACAACCAAGTCGCGCATTTGGGCGGACGGGATCGTGGTCTACGAGGCCACA 6623-6699 CTCAGTGCGCGCCAGTCGGCCATCTCGCGGAAGGGCGCAGCAGCGCGCACGGCGGCGAGCACAGTTGCGCGGCGCGC 6700-6776 AAAGTCCGCGTCAGCCATGGAGGCATTGCTATGAGCGACGGCTACAGCGACGGCTACAGCGACGGCTACAACCGGCA 6777-6853 GCCGACTGTCCGCAAAAAGCCGTGACGCGCCGAAGGCGCTCGAATCACCGGACTATCCGAACGCCACGTCGTCCGGC 6854-6930 TCGTGGCGCAGGAACGCAGCGAGTGGCTCGCCGAGCAGGCTGCACGCGCGCGAAGCATCCGCGCCTATCACGACGAC 6931-7007 GAGGGCCACTCTTGGCCGCAAACGGCCAAACATTTCGGGCTGCATCTGGACACCGTTAAGCGACTCGGCTATCGGGC 7008-7084 GAGGAAAGAGCGTGCGGCAGAACAGGAAGCGGCTCAAAAGGCCCACAACGAAGCCGACAATCCACCGCTGTTCTAAC 7085-7161 GCAATTGGGGACGGGTGTCGCGGGGGTTCCGTGGGGGGTTCCGTTGCAACGGGTCGGACAGGTAAAAGTCCTGGTAG 7162-7238 ACGCTAGTTTTCTGGTTTGGGCCATGCCTGTCTCGTTGCGTGTTTCGTTGCGCCGTTTTGAATACCAGCCAGACGAG 7239-7315 ACGGGGTTCTACGAATCTTGGTCGATACCAAGCCATTTCCGCTGAATATCGGGGAGCTCACCGCCAGAATCGGTGGT 7316-7392 TGTGGTGATGTACGTGGCGAACTCCGTTGTAGTGCCTGTGGTGGCATCCGTGGCCACTCTCGTTGCACGGTTCGTTG 7393-7469 TGCCGTTACAGGCCCCGTTGACAGCTCACCGAACGTAGTTAAAACATGCTGGTCAAACTAGGTTTACCAACGATACG 7470-7546 AGTCAGCTCATCTAGGGCCAGTTCTAGGCGTTGTTCGTTGCGCGGTTCGTTGCGCATGTTTCGTGTGGTTGCTAGAT 7547-7623 GGCTCCGCAACCACACGCTTCGAGGTTGAGTGCTTCCAGCACGGGCGCGATCCAGAAGAACTTCGTCGTGCGACTGT 7624-7700 CCTCGTTGGGATCTAGCCCGCCTAATGAGCGGGCTTTTTTTT 7701-7742

[0157] Mycobacteria overexpressing disA are attenuated for virulence. As shown in FIG. 14, when mice are infected with 3.5 log.sub.10 units by the aerosol route of either M tuberculosis harboring the pSD5B P.sub.hsp60::disA plasmid (M.tb-disA-OE or Mtb-OE) or wild type M. tuberculosis (Mtb-CDC1551), there are profound differences in the median time to death (MTD) of the animals. As can be seen, wild type M. tuberculosis (Mtb-CDC1551) gave an MTD of 150.5 days, while M. tuberculosis harboring the pSD5B P.sub.hsp60::disA plasmid (M.tb-disA-OE or Mtb-OE) was a significantly weaker pathogen giving an MTD of 321.5 days. A similar reduction in the pathogenicity is to be expected with BCG-disA-OE compared with BCG-WT. Hence, it is likely that should BCG-disA-OE be used as a cancer immunotherapy, one would anticipate reduced rates of bloodstream dissemination, reduced dysuria, reduced urgency and reduced malaise compared with BCG-WT.

[0158] Addition of CDN Cyclase Genes to rBCG other than disA

[0159] Overexpression of the PAMP immunomodulator, 3-5 c-di-GMP by overexpressing the GGDEF domain of protein BCG_RS07340. 3-5 c-di-GMP is a strong inducer of the STING-TBK1-IRF3 axis. It is produced by mycobacteria including BCG by the GGDEF domain of protein BCG_RS07340 (previously BCG_1416c) and by the M. tuberculosis Rv1354c gene. The BCG_RS07340 protein (100% identical to the M. tuberculosis Rv1354c protein) encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase. Hence the portion that functions as a diguanylate cyclase is an endogenous CDN-producing enzyme in BCG. The full-length BCG_RS07340 polypeptide is 623 amino acids in length, and its domain structure is: N-terminus-GAF-GGDEF-EAL-C-terminus as shown in FIG. 11. The GAF domain (approximately amino acids 1-190) is a regulatory domain which influences the activity of the other domains. The GGDEF domain (approximately amino acids 190-350) is a diguanylate cyclase catalyzing the reaction 2 GTP.fwdarw.c-di-GMP+2 pyrophosphates. The EAL domain (approximately amino acids 350-623) is a diguanylate phosphodiesterase catalyzing the reaction c-di-GMP.fwdarw.2 GMP. By genetically removing the DNA sequences that encode the C-terminal EAL domain, it is possible to use the DNA encoding the GGDEF domain to generate a recombinant BCG that will overexpress diguanulate cyclase activity. This may be accomplished by also deleting the DNA encoding the regulatory-sensor GAF domain and/or the use of mutations in the DNA encoding the GAF domain to relieve any cyclase inhibitory activity it may possess. Such techniques to generate constitutively active recombinant forms of the BCG_RS07340 protein will produce high levels of c-di-GMP in recombinant BCG.

[0160] SEQ ID NO: 4

[0161] Bifunctional diguanylate cyclase/phosphodiesterase BCG_RS07340 from BCG and other related mycobacteria, amino acid sequence (623 amino acids; BCG protein BCG_RS07340; NCBI Reference Sequence: NC_008769.1; Protein ID WP 003898837.1; old locus tag BCG_1416c). The identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as protein Rv1354c or MT1397, and in Mycobacterium bovis as protein Mb1389c. The EAL domain is from amino acid 354 to 623 and is underlined.

TABLE-US-00004 MCNDTATPQLEELVTTVANQLMTVDAATSAEVSQRVLAYLVEQLGVDVSF LRHNDRDRRATRLVAEWPPRLNIPDPDPLRLIYFADADPVFALCEHAKEP LVFRPEPATEDYQRLIEEARGVPVTSAAAVPLVSGEITTGLLGFIKFGDR KWHEAELNALMTIATLFAQVQARVAAEARLRYLADHDDLTGLHNRRALLQ HLDQRLAPGQPGPVAALFLDLDRLKAINDYLGHAAGDQFIHVFAQRIGDA LVGESLIARLGGDEFVLIPASPMSADAAQPLAERLRDQLKDHVAIGGEVL TRTVSIGVASGTPGQHTPSDLLRRADQAALAAKHAGGDSVAIFTADMSVS GELRNDIELHLRRGIESDALRLVYLPEVDLRTGDIVGTEALVRWQHPTRG LLAPGCFIPVAESINLAGELDRWVLRRACNEFSEWQSAGLGHDALLRINV SAGQLVTGGFVDFVADTIGQHGLDASSVCLEITENVVVQDLHTARATLAR LKEVGVHIAIDDFGTGYSAISLLQTLPIDTLKIDKTFVRQLGTNTSDLVI VRGIMTLAEGFQLDVVAEGVETEAAARILLDQRCYRAQGFLFSRPVPGEA MRHMLSARRLPPTCIPATDPALS

[0162] SEQ ID NO: 5

[0163] Bifunctional diguanylate cyclase/phosphodiesterase BCG_RS07340 from BCG and other related mycobacteria, DNA sequence (1872 nucleotides [623 codons+1 stop codons]; encodes BCG protein BCG_RS07340; NCBI Reference Sequence: NC_008769.1; Protein ID WP 003898837.1; old locus tag BCG_1416c; DNA from NC_008769.1:c1548390-1546519 Mycobacterium bovis BCG Pasteur 1173P2). The identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as protein Rv1354c or MT1397, and in Mycobacterium bovis as protein Mb1389c. EAL domain is encoded from nucleotide 1060 to 1872 and is underlined.

TABLE-US-00005 ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGT AGCCAACCAGCTCATGACAGTCGACGCTGCCACGTCAGCCGAAGTCAGTC AGCGCGTTTTGGCCTATCTAGTGGAACAGCTGGGCGTAGATGTCAGCTTT TTGCGTCATAACGATCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATG GCCACCTCGCCTCAACATACCGGACCCCGATCCGCTCAGGCTGATCTACT TCGCTGATGCCGACCCGGTGTTTGCGCTATGCGAACACGCCAAAGAGCCT CTCGTGTTCCGGCCCGAGCCGGCCACCGAGGACTATCAACGCCTCATCGA AGAAGCCCGCGGGGTTCCGGTAACGTCGGCTGCCGCCGTGCCGCTGGTAT CTGGCGAGATCACCACTGGACTGCTGGGGTTCATCAAGTTCGGTGATCGG AAATGGCACGAGGCCGAGCTTAACGCCCTCATGACCATCGCTACACTCTT CGCCCAGGTGCAGGCTCGCGTCGCCGCCGAGGCGCGGCTTCGCTATCTGG CCGACCATGACGATCTGACCGGACTGCATAACCGTCGCGCGTTGCTGCAG CACCTGGACCAAAGACTGGCCCCCGGACAACCTGGCCCGGTCGCGGCGCT ATTTCTCGACTTGGACCGCCTCAAGGCCATCAACGACTACCTGGGCCACG CCGCCGGTGACCAGTTCATCCATGTGTTCGCCCAACGGATCGGTGACGCA CTCGTTGGCGAGAGCCTGATCGCCCGACTCGGCGGCGACGAATTCGTCCT CATACCCGCATCTCCAATGAGTGCCGATGCCGCTCAACCGCTCGCCGAAC GTCTTCGCGACCAGCTCAAGGACCACGTCGCTATCGGCGGTGAGGTGCTC ACCCGCACCGTCAGTATCGGTGTCGCCTCAGGGACTCCCGGACAGCACAC ACCGTCGGACCTCCTGCGCCGAGCCGACCAAGCCGCTCTGGCAGCCAAAC ACGCCGGCGGAGATAGCGTCGCGATTTTCACCGCGGACATGTCGGTCAGC GGCGAACTGCGCAACGATATTGAACTACACCTTCGACGTGGTATCGAATC CGACGCCCTTCGCCTGGTCTACCTACCCGAGGTCGACCTACGGACCGGCG ACATTGTCGGGACCGAGGCATTGGTCCGGTGGCAGCACCCCACCCGTGGG CTGCTGGCACCGGGCTGCTTCATCCCTGTGGCCGAATCCATCAACCTTGC AGGCGAATTGGATAGATGGGTGCTGCGGAGGGCCTGCAATGAATTCTCCG AGTGGCAGTCAGCCGGTTTGGGCCACGACGCGCTGCTGCGTATCAACGTC TCAGCTGGACAGCTGGTGACGGGCGGGTTTGTTGACTTCGTCGCAGACAC GATCGGCCAGCACGGTCTGGACGCCTCGTCCGTGTGTTTGGAAATCACCG AAAACGTTGTGGTGCAAGACCTACATACCGCCAGAGCCACCCTGGCTCGA CTCAAAGAAGTCGGCGTTCACATCGCTATCGACGATTTCGGCACCGGCTA TAGCGCCATATCACTGTTGCAGACGCTACCGATCGACACGCTCAAGATCG ACAAAACATTCGTGCGGCAACTCGGAACCAACACTAGCGATCTGGTCATT GTGCGCGGCATCATGACACTCGCCGAAGGCTTCCAACTCGATGTAGTAGC CGAAGGCGTCGAGACCGAGGCTGCCGCCAGAATTCTATTGGATCAGCGCT GTTACCGTGCGCAAGGCTTCTTGTTCTCCCGGCCTGTCCCCGGGGAGGCC ATGCGGCACATGTTGTCCGCACGACGACTACCGCCGACCTGCATACCTGC AACTGACCCGGCGTTATCTTGA

[0164] SEQ ID NO: 6

[0165] Modified bifunctional diguanylate cyclase/phosphodiesterase from BCG and other related mycobacteria, with its EAL domain deleted so that it acts as a monofunctional diguanylate cyclase, amino acid sequence (353 amino acids; a fragment of BCG protein BCG_RS07340; NCBI Reference Sequence: NC_008769.1; Protein ID WP 003898837.1; old locus tag BCG_1416c). The identical sequence fragment is present in other strains of BCG, e.g., Mycobacterium tuberculosis as protein Rv1354c or MT1397, and in Mycobacterium bovis as protein Mb1389c.

TABLE-US-00006 MCNDTATPQLEELVTTVANQLMTVDAATSAEVSQRVLAYLVEQL GVDVSFLRHNDRDRRATRLVAEWPPRLNIPDPDPLRLIYFADADPVFALC EHAKEPLVFRPEPATEDYQRLIEEARGVPVTSAAAVPLVSGEITTGLLGF IKFGDRKWHEAELNALMTIATLFAQVQARVAAEARLRYLADHDDLTGLHN RRALLQHLDQRLAPGQPGPVAALFLDLDRLKAINDYLGHAAGDQFIHVFA QRIGDALVGESLIARLGGDEFVLIPASPMSADAAQPLAERLRDQLKDHVA IGGEVLTRTVSIGVASGTPGQHTPSDLLRRADQAALAAKHAGGDSVAIFT ADMSVSGEL

[0166] SEQ ID NO: 7

[0167] Modified, bifunctional diguanylate cyclase/phosphodiesterase from BCG and other related mycobacteria, with sequences encoding its EAL domain deleted so that it encodes a monofunctional diguanylate cyclase, DNA sequence (1059 nucleotides [353 codons+0 stop codons]; encodes a fragment of BCG protein BCG_RS07340; NCBI Reference Sequence: NC_008769.1; Protein ID WP 003898837.1; old locus tag BCG_1416c; DNA from NC_008769.1:c1548390-1546519 Mycobacterium bovis BCG Pasteur 1173P2). The identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as a fragment of gene Rv1354c or MT1397, and in Mycobacterium bovis as a fragment of gene Mb1389c.

TABLE-US-00007 ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGT AGCCAACCAGCTCATGACAGTCGACGCTGCCACGTCAGCCGAAGTCAGTC AGCGCGTTTTGGCCTATCTAGTGGAACAGCTGGGCGTAGATGTCAGCTTT TTGCGTCATAACGATCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATG GCCACCTCGCCTCAACATACCGGACCCCGATCCGCTCAGGCTGATCTACT TCGCTGATGCCGACCCGGTGTTTGCGCTATGCGAACACGCCAAAGAGCCT CTCGTGTTCCGGCCCGAGCCGGCCACCGAGGACTATCAACGCCTCATCGA AGAAGCCCGCGGGGTTCCGGTAACGTCGGCTGCCGCCGTGCCGCTGGTAT CTGGCGAGATCACCACTGGACTGCTGGGGTTCATCAAGTTCGGTGATCGG AAATGGCACGAGGCCGAGCTTAACGCCCTCATGACCATCGCTACACTCTT CGCCCAGGTGCAGGCTCGCGTCGCCGCCGAGGCGCGGCTTCGCTATCTGG CCGACCATGACGATCTGACCGGACTGCATAACCGTCGCGCGTTGCTGCAG CACCTGGACCAAAGACTGGCCCCCGGACAACCTGGCCCGGTCGCGGCGCT ATTTCTCGACTTGGACCGCCTCAAGGCCATCAACGACTACCTGGGCCACG CCGCCGGTGACCAGTTCATCCATGTGTTCGCCCAACGGATCGGTGACGCA CTCGTTGGCGAGAGCCTGATCGCCCGACTCGGCGGCGACGAATTCGTCCT CATACCCGCATCTCCAATGAGTGCCGATGCCGCTCAACCGCTCGCCGAAC GTCTTCGCGACCAGCTCAAGGACCACGTCGCTATCGGCGGTGAGGTGCTC ACCCGCACCGTCAGTATCGGTGTCGCCTCAGGGACTCCCGGACAGCACAC ACCGTCGGACCTCCTGCGCCGAGCCGACCAAGCCGCTCTGGCAGCCAAAC ACGCCGGCGGAGATAGCGTCGCGATTTTCACCGCGGACATGTCGGTCAGC GGCGAACTG

[0168] Overexpression of the PAMP immunomodulator, 2-5c-GAMP synthase: Q9KVG7 (Swiss-Prot). 2-5 c-GAMP is a strong inducer of the STING-TBK1-IRF3 axis. The Vibrio cholerae Q9KVG7 protein (436 amino acids) encoded by the dncV gene is a known 2-5c-GAMP synthase. It is possible to generate a recombinant dncV gene which is codon-optimized for BCG. The codon-optimized structural gene may be overexpressed in BCG by fusion to a strong promoter (such as Phsp60) or a conditionally active strong promoter such as PTET-off. Such techniques to generate a constitutively active recombinant forms of the Q9KVG7 protein will produce high levels of 2-5c-GAMP in recombinant BCG.

[0169] SEQ ID No: 8

[0170] Cyclic GMP-AMP synthase, DncV, from Vibrio cholerae, amino acid sequence (436 amino acids; UniProtKB/Swiss-Prot Protein ID Q9KVG7.1).

TABLE-US-00008 MRMTWNFHQYYTNRNDGLMGKLVLTDEEKNNLKALRKIIRLRTRDVFEEA KGIAKAVKKSALTFEIIQEKVSTTQIKHLSDSEQREVAKLIYEMDDDARD EFLGLTPRFWTQGSFQYDTLNRPFQPGQEMDIDDGTYMPMPIFESEPKIG HSLLILLVDASLKSLVAENHGWKFEAKQTCGRIKIEAEKTHIDVPMYAIP KDEFQKKQIALEANRSFVKGAIFESYVADSITDDSETYELDSENVNLALR EGDRKWINSDPKIVEDWFNDSCIRIGKHLRKVCRFMKAWRDAQWDVGGPS SISLMAATVNILDSVAHDASDLGETMKIIAKHLPSEFARGVESPDSTDEK PLFPPSYKHGPREMDIMSKLERLPEILSSAESADSKSEALKKINMAFGNR VTNSELIVLAKALPAFAQEPSSASKPEKISSTMVSG

[0171] SEQ ID No: 9

[0172] Cyclic GMP-AMP synthase, DncV, from Vibrio cholerae, DNA sequence (1311 nucleotides [436 codons+1 stop codon]; encodes UniProtKB/Swiss-Prot Protein ID Q9KVG7.1; NCBI Reference Sequence: NC_002505.1: Vibrio cholerae 01 biovar El Tor str. N16961 chromosome I, complete sequence, and nucleotides 180419-181729)

TABLE-US-00009 GTGAGAATGACTTGGAACTTTCACCAGTACTACACAAACCGAAATGATGG CTTGATGGGCAAGCTAGTTCTTACAGACGAGGAGAAGAACAATCTAAAGG CATTGCGTAAGATCATCCGCTTAAGAACACGAGATGTATTTGAAGAAGCT AAGGGTATTGCCAAGGCTGTGAAAAAAAGTGCTCTTACGTTTGAAATTAT TCAGGAAAAGGTGTCAACGACCCAAATTAAGCACCTTTCTGACAGCGAAC AACGAGAAGTGGCTAAGCTTATTTACGAGATGGATGATGATGCTCGTGAT GAGTTTTTGGGATTGACACCTCGCTTTTGGACTCAGGGAAGCTTTCAGTA TGACACGCTGAATCGCCCGTTTCAGCCTGGTCAAGAAATGGATATTGATG ATGGAACCTATATGCCAATGCCTATTTTTGAGTCAGAGCCTAAGATTGGT CATTCTTTACTAATTCTTCTTGTTGACGCGTCACTTAAGTCACTTGTAGC TGAAAATCATGGCTGGAAATTTGAAGCTAAGCAGACTTGTGGGAGGATTA AGATTGAGGCAGAGAAAACACATATTGATGTACCAATGTATGCAATCCCT AAAGATGAGTTCCAGAAAAAGCAAATAGCTTTAGAAGCAAATAGATCATT TGTTAAAGGTGCCATTTTTGAATCATATGTTGCAGATTCAATTACTGACG ATAGTGAAACTTATGAATTAGATTCAGAAAACGTAAACCTTGCTCTTCGT GAAGGTGATCGGAAGTGGATCAATAGCGACCCCAAAATAGTTGAAGATTG GTTCAACGATAGTTGTATACGTATTGGTAAACATCTTCGTAAGGTTTGTC GCTTTATGAAAGCGTGGAGAGATGCGCAGTGGGATGTTGGAGGTCCGTCA TCGATTAGTCTTATGGCTGCAACGGTAAATATTCTTGATAGCGTTGCTCA TGATGCTAGTGATCTCGGAGAAACAATGAAGATAATTGCTAAGCATTTAC CTAGTGAGTTTGCTAGGGGAGTAGAGAGCCCTGACAGTACCGATGAAAAG CCACTCTTCCCACCCTCTTATAAGCATGGCCCTCGGGAGATGGACATTAT GAGCAAACTAGAGCGTTTGCCAGAGATTCTGTCATCTGCTGAGTCAGCTG ACTCTAAGTCAGAGGCCTTGAAAAAGATTAATATGGCGTTTGGGAATCGT GTTACTAATAGCGAGCTTATTGTTTTGGCAAAGGCTTTACCGGCTTTCGC TCAAGAACCTAGTTCAGCCTCGAAACCTGAAAAAATCAGCAGCACAATGG TAAGTGGCTGA

[0173] Overexpression of the DAMP immunomodulator, 2-3 cGAMP synthase: Q8N884 (Swiss-Prot). 2-3 cGAMP is a strong inducer of the STING-TBK1-IRF3 axis. The cGAS protein is produced by the human cGAS gene to yield a 522 amino acid polypeptide which senses cytosolic DNA and functions as a 2-3 cGAMP synthase. The synthase or cyclase domain of cGAS becomes activated when cGAS binds to DNA. It is possible to generate a recombinant cGAS gene which contains only the cyclase domain and is hence constitutively active. This recombinant gene can also be codon-optimized for BCG. The codon-optimized structural gene may be overexpressed in BCG by fusion to a strong promoter (such as Phsp60) or a conditionally active strong promoter such as PTET-off. Such techniques to generate a constitutively active recombinant forms of the cGAS protein will produce high levels of 2-3c-GAMP in recombinant BCG.

[0174] SEQ ID No: 10

[0175] Cyclic 23-GMP-AMP synthase, cGAS, from Homo sapiens, amino acid sequence (522 amino acids, UniProtKB/Swiss-Prot Protein ID Q8N884.2).

TABLE-US-00010 MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGK FGPARKSGSRQKKSAPDTQERPPVRATGARAKKAPQRAQDTQPSDATSAP GAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPGLPVSA PILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCD SAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYF VKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKR KRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENK EEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNP QDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRS KEFLTKQIEYERNNEFPVFDEF

[0176] SEQ ID No: 11

[0177] Cyclic 23-GMP-AMP synthase, cGAS, from Homo sapiens, DNA sequence of mRNA with nucleotide T used in place of U (1802 nucleotides; encodes UniProtKB/Swiss-Prot Protein ID Q8N884.2; NCBI Reference Sequence: NM_138441.2. Coding sequence is 1569 nucleotides [522 codons, 1 stop codon], start codon ATG [bold underlined] at nucleotide 140; Stop codon TGA (bold, underlined) at nucleotide 1706]).

TABLE-US-00011 AGCCTGGGGTTCCCCTTCGGGTCGCAGACTCTTGTGTGCCCGCCAGTAGT GCTTGGTTTCCAACAGCTGCTGCTGGCTCTTCCTCTTGCGGCCTTTTCCT GAAACGGATTCTTCTTTCGGGGAACAGAAAGCGCCAGCCATGCAGCCTTG GCACGGAAAGGCCATGCAGAGAGCTTCCGAGGCCGGAGCCACTGCCCCCA AGGCTTCCGCACGGAATGCCAGGGGCGCCCCGATGGATCCCACCGAGTCT CCGGCTGCCCCCGAGGCCGCCCTGCCTAAGGCGGGAAAGTTCGGCCCCGC CAGGAAGTCGGGATCCCGGCAGAAAAAGAGCGCCCCGGACACCCAGGAGA GGCCGCCCGTCCGCGCAACTGGGGCCCGCGCCAAAAAGGCCCCTCAGCGC GCCCAGGACACGCAGCCGTCTGACGCCACCAGCGCCCCTGGGGCAGAGGG GCTGGAGCCTCCTGCGGCTCGGGAGCCGGCTCTTTCCAGGGCTGGTTCTT GCCGCCAGAGGGGCGCGCGCTGCTCCACGAAGCCAAGACCTCCGCCCGGG CCCTGGGACGTGCCCAGCCCCGGCCTGCCGGTCTCGGCCCCCATTCTCGT ACGGAGGGATGCGGCGCCTGGGGCCTCGAAGCTCCGGGCGGTTTTGGAGA AGTTGAAGCTCAGCCGCGATGATATCTCCACGGCGGCGGGGATGGTGAAA GGGGTTGTGGACCACCTGCTGCTCAGACTGAAGTGCGACTCCGCGTTCAG AGGCGTCGGGCTGCTGAACACCGGGAGCTACTATGAGCACGTGAAGATTT CTGCACCTAATGAATTTGATGTCATGTTTAAACTGGAAGTCCCCAGAATT CAACTAGAAGAATATTCCAACACTCGTGCATATTACTTTGTGAAATTTAA AAGAAATCCGAAAGAAAATCCTCTGAGTCAGTTTTTAGAAGGTGAAATAT TATCAGCTTCTAAGATGCTGTCAAAGTTTAGGAAAATCATTAAGGAAGAA ATTAACGACATTAAAGATACAGATGTCATCATGAAGAGGAAAAGAGGAGG GAGCCCTGCTGTAACACTTCTTATTAGTGAAAAAATATCTGTGGATATAA CCCTGGCTTTGGAATCAAAAAGTAGCTGGCCTGCTAGCACCCAAGAAGGC CTGCGCATTCAAAACTGGCTTTCAGCAAAAGTTAGGAAGCAACTACGACT AAAGCCATTTTACCTTGTACCCAAGCATGCAAAGGAAGGAAATGGTTTCC AAGAAGAAACATGGCGGCTATCCTTCTCTCACATCGAAAAGGAAATTTTG AACAATCATGGAAAATCTAAAACGTGCTGTGAAAACAAAGAAGAGAAATG TTGCAGGAAAGATTGTTTAAAACTAATGAAATACCTTTTAGAACAGCTGA AAGAAAGGTTTAAAGACAAAAAACATCTGGATAAATTCTCTTCTTATCAT GTGAAAACTGCCTTCTTTCACGTATGTACCCAGAACCCTCAAGACAGTCA GTGGGACCGCAAAGACCTGGGCCTCTGCTTTGATAACTGCGTGACATACT TTCTTCAGTGCCTCAGGACAGAAAAACTTGAGAATTATTTTATTCCTGAA TTCAATCTATTCTCTAGCAACTTAATTGACAAAAGAAGTAAGGAATTTCT GACAAAGCAAATTGAATATGAAAGAAACAATGAGTTTCCAGTTTTTGATG AATTTTGAGATTGTATTTTTAGAAAGATCTAAGAACTAGAGTCACCCTAA ATCCTGGAGAATACAAGAAAAATTTGAAAAGGGGCCAGACGCTGTGGCTC AC

[0178] SEQ ID NO: 12

[0179] Cyclic 23-GMP-AMP synthase, cGAS, from Homo sapiens with mycobacterial codon optimization, DNA sequence. (1569 nucleotides [522 codons, 1 stop codon]; encodes UniProtKB/Swiss-Prot Protein ID Q8N884.2).

TABLE-US-00012 ATGCAACCATGGCACGGGAAAGCCATGCAGCGTGCGAGCGAAGCCGGGGC GACGGCCCCCAAGGCGTCGGCGCGTAACGCGCGGGGTGCGCCCATGGACC CGACGGAGTCCCCCGCGGCGCCGGAGGCGGCCCTGCCGAAAGCGGGTAAG TTCGGTCCAGCGCGGAAAAGCGGGAGCCGCCAAAAGAAGTCCGCGCCCGA CACCCAGGAGCGTCCCCCGGTCCGGGCCACCGGCGCGCGTGCCAAAAAAG CCCCGCAACGGGCGCAAGATACGCAGCCAAGCGATGCGACCTCCGCCCCC GGGGCGGAGGGTCTGGAGCCCCCGGCCGCCCGGGAGCCAGCGCTCTCGCG CGCGGGTTCCTGCCGTCAGCGGGGCGCGCGGTGTTCCACGAAACCCCGTC CCCCACCAGGTCCCTGGGACGTGCCGTCGCCGGGTTTGCCGGTGAGCGCG CCAATCCTGGTCCGGCGCGACGCGGCCCCGGGGGCGTCGAAATTGCGTGC GGTGCTCGAGAAATTGAAGTTGTCGCGCGACGACATCTCCACGGCCGCGG GTATGGTCAAGGGCGTGGTCGATCATTTGTTGTTGCGGCTCAAGTGTGAT TCGGCGTTCCGCGGGGTGGGCTTGCTGAACACGGGGTCCTACTATGAGCA TGTCAAAATCAGCGCCCCCAACGAATTTGACGTGATGTTTAAGCTGGAAG TGCCACGTATCCAATTGGAAGAGTATTCCAATACCCGTGCGTATTATTTC GTCAAATTTAAGCGCAATCCGAAGGAAAATCCACTCAGCCAATTCTTGGA GGGCGAAATTCTGTCGGCCTCGAAAATGCTCTCCAAATTTCGTAAGATTA TCAAGGAGGAGATCAACGACATTAAGGACACGGATGTGATCATGAAACGT AAACGTGGCGGTTCCCCCGCGGTGACGCTCCTCATTTCGGAAAAAATTTC GGTGGACATTACCCTGGCGTTGGAATCGAAGTCCAGCTGGCCGGCGTCGA CCCAGGAGGGCCTGCGGATTCAAAACTGGTTGAGCGCCAAAGTGCGGAAG CAGCTGCGTCTCAAACCCTTTTATTTGGTCCCGAAACATGCCAAAGAGGG TAACGGTTTTCAAGAGGAAACCTGGCGTTTGAGCTTCTCCCACATTGAGA AGGAGATTTTGAACAACCATGGTAAGTCCAAAACGTGCTGCGAGAATAAG GAAGAAAAATGTTGTCGCAAAGATTGTCTCAAATTGATGAAATATTTGCT GGAACAACTCAAAGAGCGTTTTAAGGACAAGAAGCATCTCGACAAGTTCT CCTCGTATCACGTCAAGACCGCCTTCTTTCATGTCTGTACGCAGAACCCG CAAGATAGCCAGTGGGATCGCAAGGACTTGGGGTTGTGTTTTGACAATTG CGTCACCTATTTCTTGCAATGTTTGCGGACCGAGAAATTGGAGAACTACT TTATTCCAGAATTCAACTTGTTTTCCTCGAATCTGATTGACAAACGCTCC AAAGAGTTTCTGACGAAGCAGATTGAATACGAGCGTAACAATGAGTTTCC GGTCTTTGACGAGTTTTGA

[0180] SEQ ID NO: 13

[0181] Plasmid pMH94H-P.sub.hsp60::disA::hcGASco::mCherry which is an E. coli-mycobacterial shuttle plasmid that overexpresses the BCG disA gene, the human cGAS gene (with mycobacterial codon optimization), and mCherry from the P.sub.hsp60 promoter, DNA sequence. When introduced into BCG, M. tuberculosis, M. bovis or highly related strains, this plasmid integrates as a single copy in the mycobacterial chromosome (10842 nucleotides; promoter P.sub.hsp60 DNA comprised of a portion of the M. leprae hsp65 gene nucleotides 901 to 1068 is underlined; disA coding sequence are from nucleotides 1069 to 2145; human cGAS with mycobacterial codon optimization sequences are from nucleotides 2158 to 3726; ATG start codons and TAA or TGA stop codons are shown in boldface, underline).

TABLE-US-00013 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCC 1-83 GGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGAT 84-166 TGTACTGAGAGTGCACCAAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTT 167-249 AACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGA 250-331 ACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAA 332-414 CCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGC 415-497 TTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAG 498-580 CGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTACTATGGTTGCTTTGACGTGCGG 581-663 TGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGG 664-746 GCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAG 747-829 GGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAAATTCCGGAATT 830-912 GCACTCGCCTTAGGGGAGTGCTAAAAATGATCCTGGCACTCGCGATCAGCGAGTGCCAGGTCGGGACGGTGAGACCCAGCCAG 913-995 CAAGCTGTGGTCGTCCGTCGCGGGCACTGCACCCGGCCAGCGTAAGTAATGGGGGTTGTCGGCACCCGGTGACATGCACGCTG 996-1078 TGACTCGTCCGACCCTGCGTGAGGCTGTCGCCCGCCTAGCCCCGGGCACTGGGCTGCGGGACGGCCTGGAGCGTATCCTGCGC 1079-1161 GGCCGCACTGGTGCCCTGATCGTGCTGGGCCATGACGAGAATGTCGAGGCCATCTGCGATGGTGGCTTCTCCCTCGATGTCCG 1162-1244 CTATGCAGCAACCCGGCTACGCGAGCTGTGCAAGATGGACGGCGCCGTGGTGCTGTCCACCGACGGCAGCCGCATCGTGCGGG 1245-1327 CCAACGTGCAACTGGTACCGGATCCGTCGATCCCCACCGACGAATCGGGGACCCGGCACCGCTCGGCCGAGCGGGCCGCGATC 1228-1410 CAGACCGGTTACCCGGTGATCTCAGTGAGCCACTCGATGAACATCGTGACCGTCTACGTCCGCGGGGAACGTCACGTATTGAC 1411-1493 CGACTCGGCAACCATCCTGTCGCGGGCCAACCAGGCCATCGCAACCCTGGAGCGGTACAAAACCAGGCTCGACGAGGTCAGCC 1494-1576 GGCAACTGTCCAGGGCAGAAATCGAGGACTTCGTCACGCTGCGCGATGTGATGACGGTGGTGCAACGCCTCGAGCTGGTCCGG 1577-1659 CGAATCGGGCTGGTGATCGACTACGACGTGGTCGAACTCGGCACTGATGGTCGTCAGCTGCGGCTGCAGCTCGACGAGTTGCT 1660-1742 CGGCGGCAACGACACCGCCCGGGAATTGATCGTGCGCGATTACCACGCCAACCCGGAACCACCGTCCACGGGGCAAATCAATG 1743-1825 CCACCCTGGACGAACTGGACGCCCTGTCGGACGGCGACCTCCTCGATTTCACCGCGCTGGCAAAGGTTTTCGGATATCCGACG 1826-1908 ACCACGGAAGCGCAGGATTCGACGCTGAGCCCGCGTGGCTACCGCGCGATGGCCGGTATCCCCCGGCTCCAGTTCGCCCATGC 1909-1991 CGACCTGCTGGTCCGGGCGTTCGGAACGTTGCAGGGTCTGCTGGCGGCCAGCGCCGGCGATCTGCAATCAGTGGACGGCATCG 1992-2074 GCGCCATGTGGGCCCGTCATGTGCGCGAGGGGTTGTCACAGCTGGCGGAATCGACCATCAGCGATCAATAAGAGCACATCGAT 2075-2157 ATGCAACCATGGCACGGGAAAGCCATGCAGCGTGCGAGCGAAGCCGGGGCGACGGCCCCCAAGGCGTCGGCGCGTAACGCGCG 2158-2240 GGGTGCGCCCATGGACCCGACGGAGTCCCCCGCGGCGCCGGAGGCGGCCCTGCCGAAAGCGGGTAAGTTCGGTCCAGCGCGGA 2241-2323 AAAGCGGGAGCCGCCAAAAGAAGTCCGCGCCCGACACCCAGGAGCGTCCCCCGGTCCGGGCCACCGGCGCGCGTGCCAAAAAA 2324-2406 GCCCCGCAACGGGCGCAAGATACGCAGCCAAGCGATGCGACCTCCGCCCCCGGGGCGGAGGGTCTGGAGCCCCCGGCCGCCCG 2407-2489 GGAGCCAGCGCTCTCGCGCGCGGGTTCCTGCCGTCAGCGGGGCGCGCGGTGTTCCACGAAACCCCGTCCCCCACCAGGTCCCT 2490-2572 GGGACGTGCCGTCGCCGGGTTTGCCGGTGAGCGCGCCAATCCTGGTCCGGCGCGACGCGGCCCCGGGGGCGTCGAAATTGCGT 2573-2655 GCGGTGCTCGAGAAATTGAAGTTGTCGCGCGACGACATCTCCACGGCCGCGGGTATGGTCAAGGGCGTGGTCGATCATTTGTT 2656-2738 GTTGCGGCTCAAGTGTGATTCGGCGTTCCGCGGGGTGGGCTTGCTGAACACGGGGTCCTACTATGAGCATGTCAAAATCAGCG 2739-2821 CCCCCAACGAATTTGACGTGATGTTTAAGCTGGAAGTGCCACGTATCCAATTGGAAGAGTATTCCAATACCCGTGCGTATTAT 2822-2904 TTCGTCAAATTTAAGCGCAATCCGAAGGAAAATCCACTCAGCCAATTCTTGGAGGGCGAAATTCTGTCGGCCTCGAAAATGCT 2905-2987 CTCCAAATTTCGTAAGATTATCAAGGAGGAGATCAACGACATTAAGGACACGGATGTGATCATGAAACGTAAACGTGGCGGTT 2988-3070 CCCCCGCGGTGACGCTCCTCATTTCGGAAAAAATTTCGGTGGACATTACCCTGGCGTTGGAATCGAAGTCCAGCTGGCCGGCG 3071-3153 TCGACCCAGGAGGGCCTGCGGATTCAAAACTGGTTGAGCGCCAAAGTGCGGAAGCAGCTGCGTCTCAAACCCTTTTATTTGGTC 3154-3237 CCGAAACATGCCAAAGAGGGTAACGGTTTTCAAGAGGAAACCTGGCGTTTGAGCTTCTCCCACATTGAGAAGGAGATTTTGAAC 3238-3321 AACCATGGTAAGTCCAAAACGTGCTGCGAGAATAAGGAAGAAAAATGTTGTCGCAAAGATTGTCTCAAATTGATGAAATATTTG 3322-3405 CTGGAACAACTCAAAGAGCGTTTTAAGGACAAGAAGCATCTCGACAAGTTCTCCTCGTATCACGTCAAGACCGCCTTCTTTCAT 3406-3489 GTCTGTACGCAGAACCCGCAAGATAGCCAGTGGGATCGCAAGGACTTGGGGTTGTGTTTTGACAATTGCGTCACCTATTTCTTG 3490-3573 CAATGTTTGCGGACCGAGAAATTGGAGAACTACTTTATTCCAGAATTCAACTTGTTTTCCTCGAATCTGATTGACAAACGCTCC 3574-3657 AAAGAGTTTCTGACGAAGCAGATTGAATACGAGCGTAACAATGAGTTTCCGGTCTTTGACGAGTTTTGAAAGCTTGAGATGGTG 3658-3741 AGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCAC 3742-3825 GAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCC 3826-3909 CTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGAC 3919-3993 TACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAG 3994-4077 GACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAG 4078-4161 AAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTG 4162-4225 AAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTAC 4226-4329 AACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCAC 4330-4413 TCCACCGGCGGCATGGACGAGCTGTACAAGTAGACTAGTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGA 4414-4497 ACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAA 4498-4581 CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCG 4582-4665 GGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAA 4667-4749 GCAGAAGGCCATCCTGACGGATGGCCTTTTTTCTAGAGTCGACCACCAAGGGCACCATCTCTGCTTGGGCCACCCCGTTGGCCG 4750-4833 CAGCCAGCTCGCTGAGAGCCGTGAACGACAGGGCGAACGCCAGCCCGCCGACGGCGAGGGTTCCGACCGCTGCAACTCCCGGTG 4834-4917 CAACCTTGTCCCGGTCTATTCTCTTCACTGCACCAGCTCCAATCTGGTGTGAATGCCCCTCGTCTGTTCGCGCAGGCGGGGGGC 4918-5001 TCTATTCGTTTGTCAGCATCGAAAGTAGCCAGATCAGGGATGCGTTGCAACCGCGTATGCCCAGGTCAGAAGAGTCGCACAAGA 5002-5085 GTTGCAGACCCCTGGAAAGAAAAATGGCCAGAGGGCGAAAACACCCTCTGACCAGCGGAGCGGGCGACGGGAATCGAACCCGCG 5086-5169 TAGCTAGTTTGGAAGAATGGGTGTCTGCCGACCACATATGGGCCGGTCAAGATAGGTTTTTACCCCCTCTCGGCTGCATCCTCT 5170-5253 AAGTGGAAAGAAATTGCAGGTCGTAGAAGCGCGTTGAAGCCTGAGAGTTGCACAGGAGTTGCAACCCGGTAGCCTTGTTCACGA 5254-5337 CGAGAGGAGACCTAGTTGGCACGTCGCGGATGGGGATCGCTGAAGACTCAGCGCAGCGGGAGGATCCAAGCCTCATACGTCAAC 5338-5421 CCGCAGGACGGTGTGAGGTACTACGCGCTGCAGACCTACGACAACAAGATGGACGCCGAAGCCTGGCTCGCGGGCGAGAAGCGG 5422-5505 CTCATCGAGATGGAGACCTGGACCCCTCCACAGGACCGGGCGAAGAAGGCAGCCGCCAGCGCCATCACGCTGGAGGAGTACACC 5506-5589 CGGAAGTGGCTCGTGGAGCGCGACCTCGCAGACGGCACCAGGGATCTGTACAGCGGGCACGCGGAGCGCCGCATCTACCCGGTG 5590-5673 CTAGGTGAAGTGGCGGTCACAGAGATGACGCCAGCTCTGGTGCGTGCGTGGTGGGCCGGGATGGGTAGGAAGCACCCGACTGCC 5674-5757 CGCCGGCATGCCTACAACGTCCTCCGGGCGGTGATGAACACAGCGGTCGAGGACAAGCTGATCGCAGAGAACCCGTGCCGGATC 5768-5841 GAGCAGAAGGCAGCCGATGAGCGCGACGTAGAGGCGCTGACGCCTGAGGAGCTGGACATCGTCGCCGCTGAGATCTTCGAGCAC 5842-5925 TACCGGATCGCGGCATACATCCTGGCGTGGACGAGCCTCCGGTTCGGAGAGCTGATCGAGCTTCGCCGCAAGGACATCGTGGAC 5926-6009 GACGGCATGACGATGAAGCTCCGGGTGCGCCGTGGCGCTTCCCGCGTGGGGAACAAGATCGTCGTTGGCAACGCCAAGACCGTC 6010-6093 CGGTCGAAGCGTCCTGTGACGGTTCCGCCTCACGTCGCGGAGATGATCCGAGCGCACATGAAGGACCGTACGAAGATGAACAAG 6094-6177 GGCCCCGAGGCATTCCTGGTGACCACGACGCAGGGCAACCGGCTGTCGAAGTCCGCGTTCACCAAGTCGCTGAAGCGTGGCTAC 6178-6261 GCCAAGATCGGTCGGCCGGAACTCCGCATCCACGACCTCCGCGCTGTCGGCGCTACGTTCGCCGCTCAGGCAGGTGCGACGACC 6262-6345 AAGGAGCTGATGGCCCGTCTCGGTCACACGACTCCTAGGATGGCGATGAAGTACCAGATGGCGTCTGAGGCCCGCGACGAGGCT 6346-6429 ATCGCTGAGGCGATGTCCAAGCTGGCCAAGACCTCCTGAAACGCAAAAAGCCCCCCTCCCAAGGACACTGAGTCCTAAAGAGGG 6430-6513 GGGTTTCTTGTCAGTACGCGAAGAACCACGCCTGGCCGCGAGCGCCAGCACCGCCGCTCTGTGCGGAGACCTGGGCACCAGCCC 6514-6597 CGCCGCCGCCAGGAGCATTGCCGTTCCCGCCAGCTGAGTTCTGTTGTGCGCCGCCTATGTAGAGCTGGTCGTTGTAGGTCCGA 6598-6680 TCTCCAGGCGACTTTCCGGCGACGCTGAGGATGTCGATCACAGAGCCTCCGGGACCGCCGGTTGCGGTCAAACCTGACCATCC 6681-6763 GACAGCGGACGCCGTGGTGTTTCCTCCAGGGCCTCCGGCCTTGCCTGAGAATACAGAGCCAGCTCCCGCTGCGCCTCCAGCTC 6764-6846 CGACGAGCCCGGTGATCGTCTTGGTCGACCTGCAGGCATGCAAAAGCTGATCCTTGCCGAGCTGGGATGGAAGCCCGGCCGAC 6847-6929 CCACCCTGGAGGAGATGATCGAGGATGCCAGGGCCTTTCACGCCCGCCGCTGCTGAGCGTCCGCCGCCGGGCCCGCACCGCCG 6830-7012 TCGGCCGGCCCGCTCCGGGCTCGCAGCAGCGGGCTTCGGCGCGGGCCCGGGGCTCCCGGGCCGCCGGGCGGGGCTCCGCCCGG 7013-7095 CGGCCGCCGGGGGCCGGGGGCGGCGCCGGGCGGCCCGGGGCGTCAGGCGCCGGGGGCGGTGTCCGGCGGCCCCCAGAGGAACT 7096-7178 GCGCCAGTTCCTCCGGATCGGTGAAGCCGGAGAGATCCAGCGGGGTCTCCTCGAACACCTCGAAGTCGTGCAGGAAGGTGAAG 7179-7261 GCGAGCAGTTCGCGGGCGAAGTCCTCGGTCCGCTTCCACTGCGCCCCGTCGAGCAGCGCGGCCAGGATCTCGCGGTCGCCCCG 7262-7344 GAAGGCGTTGAGATGCAGTTGCACCAGGCTGTAGCGGGAGTCTCCCGCATAGACGTCGGTGAAGTCGACGATCCCGGTGACCT 7345-7427 CGGTCGCGGCCAGGTCCACGAAGATGTTGGTCCCGTGCAGGTCGCCGTGGACGAACCGGGGTTCGCGGCCGGCCAGCAGCGTG 7428-7510 TCCACGTCCGGCAGCCAGTCCTCCAGGCGGTCCAGCAGCCGGGGCGAGAGGTAGCCCCACCCGCGGTGGTCCTCGACGGTCGC 7511-7593 CGCGCGGCGTTCCCGCAGCAGTTCCGGGAAGACCTCGGAATGGGGGGTGAGCACGGTGTTCCCGGTCAGCGGCACCCTGTGCA 7594-7676 GCCGGCCGAGCACCCGGCCGAGTTCGCGGGCCAGGGCGAGCAGCGCGTTCCGGTCGGTCGTGCCGTCCATCGCGGACCGCCAG 7677-7759 GTGGTGCCGGTCATCCGGCTCATCACCAGGTAGGGCCACGGCCAGGCTCCGGTGCCGGGCCGCAGCTCGCCGCGGCCGAGGAG 7760-7842 GCGGGGCACCGGCACCGGGGCGTCCGCCAGGACCGCGTACGCCTCCGACTCCGACGCGAGGCTCTCCGGACCGCACCAGTGCT 7743-7925 CGCCGAACAGCTTGATCACCGGGCCGGGCTCGCCGACCAGTACGGGGTTGGTGCTCTCGCCGGGCACCCGCAGCACCGGCGGC 7926-8008 ACCGGCAGCCCGAGCTCCTCCAGGGCTCGGCGGGCCAGCGGCTCCCAGAATTCCTGGTCGTTCCGCAGGCTCGCGTAGGAATC 8009-8091 ATCCGAATCAATACGGTCGAGAAGTAACAGGGATTCTTGTGTCACAGCGGACCTCTATTCACAGGGTACGGGCCGGCTTAATT 8092-8174 CCGCACGGCCGGTCGCGACACGGCCTGTCCGCACCGCGGATCAGGCGTTGACGATGACGGGCTGGTCGGCCACGTCGGGGACG 8175-8257 TTCTCGGTGGTGCTGCGGTCGGGATCGCCAATCTCTACGGGCCGACCGAGGCGACGGTGTACGCCACCGCCTGGTTCTGCGAC 8258-8340 GGCGAGGCGCCGTCCCAGGCCCCGCCGATCCCCGTCCCCCGCGTCGTCGAGCGCGGTGCCGACGACACCGCCGCGTGGCTCGT 8341-8423 CACGGAGGCCGTCCCCGGCGTCGCGGCGGCCGAGGAGTGGCCCGAGCACCAGCGGTTCGCCGTGGTCGAGGCGATGGCGGAGC 8424-8506 TGGCCCGCGCCCTCCACGAGCTGCCCGTGGAGGACTGCCCCTTCGACCGGCGCCTCGACGCGGCGGTCGCCGAGGCCCGGCGG 8507-8589 AACGTCGCCGAGGGCCTGTGGACCTCGACGACCTGCAGGCATGCAAGCTAGCTTTTGTTATCCGCTCACAATTCCACACAACA 8590-8672 TACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCC 8673-8755 GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG 8756-8838 CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAA 8839-8921 TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG 8922-9004 GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA 9005-9087 CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG 9088-9170 GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC 9171-9253 GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTC 9254-9336 CAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA 9337-9419 CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACC 9420-9502 TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT 9503-9585 TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTT 9586-9668 AAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA 9669-9751 AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTC 8752-9834 ATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC 9835-9917 CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA 9918-10000 ACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT 10001-10085 GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA 10086-10169 GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTG 10170-10253 TTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTAC 10254-10337 TCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACAT 10338-10421 AGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT 10422-10505 TCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG 10506-10589 CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATT 10590-10673 TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC 10674-10757 CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGT 10758-10841 C 10842-10842

[0182] Knocking out Endogenous BCG Phosphodiesterase Genes and Intragenic Segments Encoding Phosphodiesterase Domains in Order to Increase CDN PAMP and DAMP Levels

[0183] Overexpression of CDNs by knocking out an endogenous BCG phosphodiesterase: WP_003414507

[0184] The BCG AHM08589.1 protein encodes a 316 amino acid endogenous bifunctional c-di-AMP and cGAMP phosphodiesterase in BCG that is 100% identical to the M tuberculosis Rv2837c over the C-terminal 316 amino acids (also called CdnP, CnpB, 3-to-5 oligoribonuclease A, bifunctional oligoribonuclease, or PAP phosphatase NrnA). The M. tuberculosis Rv2837c protein is known to hydrolyze both 3-5 c-di-AMP (bacterial PAMP molecule) and 2-3cGAMP (host DAMP molecule). Since the BCG protein is 100% identical over the C-terminal 315 amino acids, knockout (gene replacement) of the BCG AHM08589.1 protein will lead to increased levels of CDNs (3-5 c-di-AMP and 2-3cGAMP) in recombinant BCG.

[0185] SEQ ID No: 14

[0186] Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP (also called CnpB, 3-to-5 oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA) from BCG, amino acid sequence (316 amino acids; BCG protein AHM08589.1; NCBI Reference DNA Sequence: CP003494.1 from BCG strain ATCC 35743; NCBI Reference Protein Identifier WP_003414507). A similar sequence is present in Mycobacterium tuberculosis as protein Rv2837c or MT2903, and in Mycobacterium bovis as protein Mb2862c.

TABLE-US-00014 MDAVGAAALLSAAARVGVVCHVHPDADTIGAGLALALVLDGCGKRVEVSF AAPATLPESLRSLPGCHLLVRPEVMRRDVDLVVTVDIPSVDRLGALGDLT DSGRELLVIDHHASNDLFGTANFIDPSADSTTTMVAEILDAWGKPIDPRV AHCIYAGLATDTGSFRWASVRGYRLAARLVEIGVDNATVSRTLMDSHPFT WLPLLSRVLGSAQLVSEAVGGRGLVYVVVDNREWVAARSEEVESIVDIVR TTQQAEVAAVFKEVEPHRWSVSMRAKTVNLAAVASGFGGGGHRLAAGYTT TGSIDDAVASLRAALG

[0187] SEQ ID No: 15

[0188] Bifunctional c-di-AMP and cGAMP phosphodiesterase gene, cdnP (also called cnpB or gene for 3-to-5 oligoribonuclease A, bifunctional oligoribonuclease, or PAP phosphatase NrnA) from BCG, DNA sequence (951 nucleotides [316 codons and 1 stop codon]; encodes BCG protein AHM08589.1; NCBI Reference Sequence: CP003494.1 from BCG strain ATCC 35743). A similar sequence is present in Mycobacterium tuberculosis encoding protein Rv2837c or MT2903, and in Mycobacterium bovis encoding protein Mb2862c.

TABLE-US-00015 GTGGACGCCGTCGGTGCCGCTGCGCTGTTGTCGGCCGCTGCCAGGGTCGG GGTAGTCTGCCACGTCCACCCCGATGCCGACACCATCGGCGCCGGATTGG CATTGGCATTGGTGTTGGACGGGTGCGGCAAGCGGGTAGAGGTCAGCTTT GCCGCGCCGGCGACACTGCCCGAGTCGCTGCGTTCGCTGCCGGGCTGCCA TCTGCTGGTCCGCCCTGAGGTGATGCGCCGCGATGTCGATTTGGTTGTGA CTGTTGACATTCCGAGTGTTGATCGGCTCGGTGCTCTGGGCGATCTAACT GATTCCGGGCGGGAGCTCCTGGTAATCGACCATCACGCCTCCAACGACCT GTTCGGCACCGCGAATTTCATTGACCCGTCGGCGGATTCCACCACGACGA TGGTTGCCGAGATCCTCGACGCGTGGGGGAAACCGATAGACCCGCGCGTC GCGCACTGCATCTACGCCGGGTTGGCGACCGACACGGGGTCGTTTCGCTG GGCCAGTGTGCGGGGGTATCGGCTGGCGGCGCGGCTGGTAGAGATCGGTG TGGACAACGCCACCGTCAGCAGGACCTTGATGGACAGCCATCCCTTCACC CTGGTTGCCGTTGTATCGCGGGTGTTGGGTTCGGCGCAGCTGGTGTCCGA GGCGGTCGGTGGCCGCGGGCTGGTTTACGTCGTCGTCGACAACCGGGAGT GGGTCGCTGCGCGCTCGGAGGAAGTGGAAAGCATCGTCGACATCGTCCGC ACCACGCAACAAGCCGAGGTCGCGGCGGTGTTCAAGGAGGTCGAACCGCA TCGGTGGTCGGTGTCGATGCGGGCTAAGACCGTGAATTTGGCCGCGGTTG CCTCTGGGTTCGGTGGCGGTGGTCACCGGCTGGCCGCGGGGTATACGACC ACCGGCTCGATCGACGACGCTGTGGCGTCGTTGCGCGCGGCGCTTGGTTA G

[0189] SEQ ID No: 16

[0190] Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP (also called CnpB, Rv2837c, or MT2903, 3-to-5 oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA) from Mycobacterium tuberculosis, amino acid sequence (336 amino acids; M. tuberculosis protein WP_003905944.1; NCBI/GenBank Reference Sequence: AL123456 from M. tuberculosis strain H37Rv). The M. tuberculosis protein has 20 additional amino acids at its N-terminus compared with the BCG protein (SEQ ID No: 14) which are underlined and boldfaced.

TABLE-US-00016 MTTIDPRSELVDGRRRAGARVDAVGAAALLSAAARVGVVCHVHPDADTIG AGLALALVLDGCGKRVEVSFAAPATLPESLRSLPGCHLLVRPEVMRRDVD LVVTVDIPSVDRLGALGDLTDSGRELLVIDHHASNDLFGTANFIDPSADS TTTMVAEILDAWGKPIDPRVAHCIYAGLATDTGSFRWASVRGYRLAARLV EIGVDNATVSRTLMDSHPFTWLPLLSRVLGSAQLVSEAVGGRGLVYVVVD NREWVAARSEEVESIVDIVRTTQQAEVAAVFKEVEPHRWSVSMRAKTVNL AAVASGFGGGGHRLAAGYTTTGSIDDAVASLRAALG

[0191] Overexpression of CDNs by knocking out an endogenous BCG phosphodiesterase domain: EAL domain of protein BCG_RS07340 (previously BCG_1416c). The BCG_RS07340 protein (SEQ ID No: 4) is encoded by the DNA sequence shown in SEQ ID No: 5. The BCG_RS07340 protein is 100% identical to the M. tuberculosis Rv1354c protein and is an endogenous CDN PDE in BCG. The full-length polypeptide is 623 amino acids in length, and it encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase. The domain structure is: N-terminus-GAF-GGDEF-EAL-C-terminus as shown in FIG. 11. The GAF domain (approximately amino acids 1-190) is a regulatory domain which influences the activity of the other domains. The GGDEF domain (approximately amino acids 190-350) is a diguanylate cyclase catalyzing the reaction 2 GTP.fwdarw.4 c-di-GMP +2 pyrophosphates. The EAL domain (amino acids 354 to 623, highlighted in SEQ ID No: 4) is a diguanylate phosphodiesterase catalyzing the reaction c-di-GMP.fwdarw.4 2 GMP. As the EAL domain of this protein is known to cleave 3-5 c-di-GMP, knockout of this endogenous cyclic dinucleotide phosphodiesterase domain will increase the levels of c-di-GMP produced by BCG. Targeted knockout of the EAL domain may be accomplished by gene replacement of the full-length WT BCG_RS07340 gene with one which encodes only amino acids 1-353 (the GAF-GGDEF domains), that is truncating the coding sequence of the gene to exclude the sequences that encode amino acids 354-623 (shown as the underlined DNA sequence in SEQ ID No: 5) and including an appropriate stop codon and transcription termination sequence. Recombinant BCG lacking the EAL domain of BCG_RS07340 will lead to increased levels of the CDN PAMP c-di-GMP.

[0192] Overexpression of CDNs by knocking out an endogenous BCG phosphodiesterase: BCG AHM07112. The BCG_AHM07112 protein is an endogenous diguanylate phosphodiesterase in BCG (homologous the 307 amino acid M. tuberculosis Rv1357c protein). Some strains of BCG lack BCG AHM07112 altogether while others such as BCG Tice harbor it. Among the BCG strains that have this polypeptide, the protein may be 288 amino acids in length (such as in BCG ATCC 35743) or 307 amino acids in length (such as in BCG Pasteur 1173 P2). The BCG_AHM07112 protein from BCG ATCC 35743 is 288 amino acids in length and is 100% identical to the M tuberculosis Rv1357c protein over its C-terminal 287 amino acids. The domain structure of BCG AHM07112 is that of a single EAL domain (FIG. 11). As the M. tuberculosis Rv1357c protein is known to cleave 3-5 c-di-GMP, it is highly likely that the BCG protein performs the same reaction. Knockout of this endogenous cyclic dinucleotide phosphodiesterase in BCG is anticipated to increase the levels of c-di-GMP produced by BCG. Targeted knockout of the EAL domain may be accomplished by gene replacement of the full-length WT BCG AHM07112 gene and subsequent generation of an unmarked deletion.

[0193] SEQ ID No: 17

[0194] Diguanylate phosphodiesterase AHM07112.1 from BCG and other related mycobacteria, amino acid sequence (288 amino acids; GenBank Reference Sequence: CP003494.1; from BCG strain ATCC 35743). AHM07112.1 is 100% identical to the C-terminal 287 amino acids of the diguanylate phosphodiesterase of Mycobacterium tuberculosis protein Rv1357c or MT1400 and of Mycobacterium bovis as protein Mb1392c.

TABLE-US-00017 MIDYEEMFRGAMQARAMVANPDQWADSDRDQVNTRHYLSTSMRVALDRGE FFLVYQPIIRLADNRIIGAEALLRWEHPTLGTLLPGRFIDRAENNGLMVP LTAFVLEQACRHVRSWRDHSTDPQPFVSVNVSASTICDPGFLVLVEGVLG ETGLPAHALQLELAEDARLSRDEKAVTRLQELSALGVGIAIDDFGIGFSS LAYLPRLPVDVVKLGGKFIECLDGDIQARLANEQITRAMIDLGDKLGITV TAKLVESPSQAARLRAFGCKAAQGWHFAKALPVDFFRE

[0195] SEQ No: 18

[0196] Diguanylate phosphodiesterase AHM07112.1 from BCG and other related mycobacteria, DNA sequence (867 nucleotides [288 codons, 1 stop codon]; GenBank Reference Sequence: CP003494.1; from BCG strain ATCC 35743). AHM07112.1 is 100% identical to the C-terminal 287 amino acids of the diguanylate phosphodiesterase of Mycobacterium tuberculosis protein Rv1357c or MT1400 and of Mycobacterium bovis as protein Mb1392c.

TABLE-US-00018 1 ttgatcgactacgaagagatgtttaggggcgcgatgcaagcgcgagcgatggtagccaat 61 cctgaccaatgggcggactccgaccgcgaccaggtcaacactcgccattatctgtccact 121 tcgatgcgcgtggcactggatcgcggtgaattcttcctcgtctaccagccaatcatccgg 181 cttgccgacaaccgcatcatcggcgccgaggccctgctgcgctgggaacacccgacgttg 241 ggcacgctactcccgggccggttcatcgaccgtgccgagaacaacggactgatggtgccg 301 ctcacggccttcgtgctcgagcaggcctgccgccacgtccgcagttggcgtgaccacagc 361 accgacccgcaaccgtttgtcagcgtcaacgtctccgccagcaccatctgcgatcccggc 421 ttcctggtgctggtcgaaggtgtgctcggcgaaaccggcctgcccgcccatgccctgcag 481 ctcgaactggccgaggacgcgcgccttagcagagacgagaaggcggtgaccaggctacaa 541 gaattgtccgctctcggcgtcggcatcgccatcgacgacttcggcattggattctccagc 601 ctcgcctaccttccccgcctccccgtcgacgtggtcaaactcgggggaaagttcatcgag 661 tgcctcgatggcgacattcaagctcggctggccaacgaacagatcacccgggcaatgatc 721 gaccttggcgacaagctcggtatcaccgtcactgcaaagctagtcgaaagccccagccaa 781 gccgcccggttgcgcgccttcggctgtaaagccgcacaaggctggcactttgccaaggca 841 ctgccggtcgactttttcagagagtag

[0197] SEQ ID No: 19

[0198] Diguanylate phosphodiesterase Rv1357c or MT1400 from Mycobacterium tuberculosis and BCG Pasteur 1173 P2, amino acid sequence (307 amino acids. NCBI/GenBank Reference Sequence: AL123456 from M. tuberculosis strain H37Rv). The 19 amino acid N-terminal extension is present in the M. tuberculosis and in BCG Pasteur strain 1173 P2 but absent in several other BCG strains. The 19 amino acid N-terminal extension is underlined and boldfaced. The C-terminal 287 amino acids of M. tuberculosis Rv1357c are 100% identical to the BCG diguanylate phosphodiesterase AHM07112.1.

TABLE-US-00019 MDRCCQRATAFACALRPTKLIDYEEMFRGAMQARAMVANPDQWADSDRDQ VNTRHYLSTSMRVALDRGEFFLVYQPIIRLADNRIIGAEALLRWEHPTLG TLLPGRFIDRAENNGLMVPLTAFVLEQACRHVRSWRDHSTDPQPFVSVNV SASTICDPGFLVLVEGVLGETGLPAHALQLELAEDARLSRDEKAVTRLQE LSALGVGIAIDDFGIGFSSLAYLPRLPVDVVKLGGKFIECLDGDIQARLA NEQITRAMIDLGDKLGITVTAKLVETPSQAARLRAFGCKAAQGWHFAKAL PVDFFRE

[0199] The sequences referenced in the application are summarized in Table 1 below.

TABLE-US-00020 TABLE 1 SEQUENCE NUMBER DESCRIPTION SEQ ID NO: 1 Diadenylate cyclase DisA from BCG and other related mycobacteria, amino acid sequence SEQ ID NO: 2 Diadenylate cyclase disA from BCG and other related mycobacteria, DNA sequence SEQ ID NO: 3 Plasmid pSD5B-P.sub.hsp60::disA which overexpresses the disA gene, DNA sequence SEQ ID NO: 4 Bifunctional diguanylate cyclase/phosphodiesterase BCG_RS07340 from BCG and other related mycobacteria, amino acid sequence SEQ ID NO: 5 Bifunctional diguanylate cyclase/phosphodiesterase BCG_RS07340 from BCG and other related mycobacteria, DNA sequence SEQ ID NO: 6 Modified, bifunctional diguanylate cyclase/phosphodiesterase from BCG and other related mycobacteria lacking the EAL domain so that it functions as a monofunctional diguanylate cyclase, amino acid sequence SEQ ID NO: 7 Modified, bifunctional diguanylate cyclase/phosphodiesterase from BCG and other related mycobacteria lacking the EAL domain so that it functions as a monofunctional diguanylate cyclase, DNA sequence SEQ ID NO: 8 Cyclic GMP-AMP synthase DncV from Vibrio cholerae, amino acid sequence SEQ ID NO: 9 Cyclic GMP-AMP synthase dncV from Vibrio cholerae, DNA sequence SEQ ID NO: 10 Cyclic GMP-AMP synthase cGAS from Homo sapiens, amino acid sequence SEQ ID NO: 11 Cyclic GMP-AMP synthase cGAS from Homo sapiens, DNA sequence SEQ ID NO: 12 Cyclic GMP-AMP synthase cGAS gene from Homo sapiens with mycobacterial codon optimization, DNA sequence SEQ ID NO: 13 Plasmid pMH94H- P.sub.hsp60::disA::COcGAS::mCherry which overexpresses the disA gene, the codon-optimized human cGAS gene, and mCherry, DNA sequence SEQ ID NO: 14 Bifunctional c-di-AMP & cGAMP phosphodiesterase CdnP from BCG, amino acid sequence SEQ ID NO: 15 Bifunctional c-di-AMP & cGAMP phosphodiesterase CdnP from BCG, DNA sequence SEQ ID NO: 16 Bifunctional c-di-AMP & cGAMP phosphodiesterase CdnP from M. tuberculosis with 20 amino acid N-terminal extension, amino acid sequence. SEQ ID NO: 17 Diguanylate phosphodiesterase AHM07112.1 from BCG and other related mycobacteria, amino acid sequence SEQ ID NO: 18 Diguanyiate phosphodiesterase AHM07112.1 from BCG and other related mycobacteria, DNA sequence SEQ ID NO: 19 Diguanyiate phosphodiesterase Rv1357c or MT1400 from Mycobacterium tuberculosis and BCG Pasteur 1173 P2 with 19 amino acid N-terminal extension, amino acid sequence

[0200] In some embodiments, the present invention relates to an expression cassette or expression vector comprising a nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof, a nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof, a nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; or a combination thereof. In some embodiments, the expression vector or expression cassette further comprises a nucleic acid sequence encoding a DNA integrity scanning (disA) protein which functions as a diadenylate cyclase, or a functional part thereof. In some embodiments, the nucleic acid sequence encoding a Rv1354c protein does not contain a phosphodiesterase gene or phosphodiesterase domain. In some embodiments, the expression vector or expression cassette does not contain a phosphodiesterase gene or phosphodiesterase domain.

[0201] Methods for generating expression vectors and expression cassettes, transforming Mycobacteria and isolating the same have been described. In some embodiments, an expression vector or expression cassette of the invention comprises one or more regulatory sequences, e.g., a promoter and/or enhancer element, operably linked to a nucleic acid of the invention which controls or influences transcription of the nucleic acid. In some embodiments, an expression vector or expression cassette of the invention comprises one or more sequences operably linked to a nucleic acid of the invention which directs termination of transcription, post-transcriptional cleavage, and/or polyadenylation. In some embodiments, an expression vector or expression cassette of the invention comprises a variable length intervening sequence and/or a selectable marker gene operably linked to a nucleic acid of the invention.

[0202] In some embodiments, the present invention relates to a strain of Mycobacterium comprising an expression vector or expression cassette of the invention described herein. In some embodiments, the strain of Mycobacterium is Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof. In some embodiments, the strain of Mycobacterium is BCG. In some embodiments, the strain comprises the plasmid of SEQ ID NO: 13.

[0203] In some embodiments, the present invention relates to a strain of Mycobacterium that expresses or overexpresses diadenylate cyclase and/or expresses or overexpresses one or more other cyclase genes or domains (e.g., those described herein). In some embodiments, the expression or overexpression results in release of one or more STING agonists (e.g., c-di-AMP, c-di-GMP, 2-3 cGAMP, and/or 3-3 cGAMP). In some embodiments, the present invention relates to a strain of Mycobacterium that expresses or overexpresses diadenylate cyclase and/or does not express a phosphodiesterase (PDE) that hydrolyzes STING agonists (e.g., contains a deletion of a PDE gene that hydrolyzes STING agonists). See, e.g., FIG. 12. In some embodiments, the strain of Mycobacterium is Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof. In some embodiments, the strain of Mycobacterium is BCG.

Statistically Significant Anti-Tumor Effects with BCG-disA-OE in the Rat MNU Bladder Cancer Model

[0204] The rat MNU bladder cancer model is a validated model of bladder cancer in which administration of intravesical BCG can be shown to be therapeutic (FIG. 6 and Kates et al. PMID 28588015). The inventors extended their previous findings of the therapeutic effect of BCG-disA-OE versus BCG-WT which were shown in FIG. 7. The inventors have now performed the 16 week rat MNU model twice. FIG. 7 was based on Expt 1 and shows that BCG-disA-OE displays a trend towards a better outcome versus BCG-WT. After performing Expt 2 and combining its data with Expt 1, the inventor now show that BCG-disA-OE is statistically significantly superior to no treatment (p =0.048) whereas BCG-WT is not statistically significantly superior to no treatment (data shown in FIG. 17).

Reduction of Tumor-Suppressive Treg Cells by BCG-disA-OE in a Murine Syngeneic Bladder Cancer Tumor Model.

[0205] In the MNU rat bladder cancer model the amount of bladder tissue at the end of the 16 week experiment is insufficient to perform flow cytometry. In order to study the cell population changes elicited by BCG-disA-OE the inventors developed a murine syngeneic bladder cancer tumor model using BBN975 cells. The model allows for large tumors (>1.5 cm in diameter) to develop on the mouse flank. Mice were treated with BCG-disA-OE and BCG-WT by intratumoral injection. As is shown in FIG. 18, the use of BCG-disA-OE led to reduced levels of tumor-associated CD4+ Treg cells, tumor-associated CD8+ Treg cells, and splenic CD4+ Treg cells.

BCG-disA-OE Delivers Sustained STING agonist from the Intracellular Compartment. Persistence of BCG in the Bladder.

[0206] Bowyer et al (The persistence of bacilli Calmette-Guerin in the bladder after intravesical treatment for bladder cancer. Brit J Urol. 1995; 75: 188-192. PMID 7850324) evaluated 125 bladder cancer patients from 1986-1992 who received intravesical BCG. Patients were asked to provide monthly urine samples which were then sent for mycobacterial culture. 90 patients survived and were compliant with the monthly urine samples. 4/90 patients (4.4%) had persistent BCG in their urine, one for up to 16.5 months. A fifth patient required a cystectomy 7 weeks after completing intravesical BCG treatments and was found to have microscopic evidence of acid-fast bacilli in the bladder by microscopy.

[0207] Durek et al. (The fate of bacillus Calmette-Guerin after intravesical instillation. J Urol. 2001; 165: 1765-1768. PMID 11342972) studied 49 patients with serial urine cultures following intravesical BCG. BCG was in the urine detected in 96.4% of the specimens after 2 hours and in 67.9% after 24 hours after instillation. The number of positive specimens decreased and it was 27.1% on day 7 immediately before the next instillation (FIG. 44). The investigators also evaluated bladder biopsies by PCR for mycobacterial DNA within 1 week after the 6.sup.th instillation (instillations were given monthly). In 14 of 44 bladder biopsies (31.8%) mycobacterial ribosomal DNA was found. Additionally, positive PCRs for mycobacterial DNA was evident up to 24 months in between 4.2% and 37.5% of the investigated biopsies.

[0208] The fact that BCG is known to persist in bladder tissue represents an important advantage of the BCG-disA-OE strategy for STING agonist deliver in cancer. While numerous technologies have focus on generating small molecule STING agonists, such agents have relatively short exposure times. In contrast, as an intracellular microorganisms and as demonstrated by the Bowyer and Durek studies, BCG persists in cells and tissues for many weeks. The persistence of BCG-disA-OE in tissue offers sustained long-term deliver of the STING agonist in the tumor microenvironment

BCG-disA-OE is Safer than BCG-WT in Two Separate Mouse Models

[0209] Intravesical BCG treatment in humans is associated with dysuria, fatigue, and malaise in treated patients. Additional more severe adverse effects are persistent cystitis with BCG and disseminated BCGosis. The patient safety of BCG was reviewed extensively in O'Donnell et al (Up-to-date, 2019). The incidence of dissemination of BCG into the bloodstream after intravesical instillation is estimated at 1/15,000 patients. To test the safety of BCG-disA-OE compared to BCG-WT the inventors used two mouse models of BCG infection where the BCG strains were aerosolized into the lungs of immunocompetent BABL/c mice or immunosuppressed SCID mice. As shown in FIG. 19, BCG-disA-OE was less capable of proliferating in immunocompetent mouse lungs than BCG-WT, and it was less lethal in a time-to-death assay in immunosuppressed mice.

BCG has been Shown to Elicit Trained Immunity which has been Associated with its Therapeutic Benefit in Solid and Liquid Tumors and for Diabetes. STING agonist Overexpressing BCG Strains Elicit Stronger Trained Immunity Changes than BCG-WT

[0210] Trained immunity. Trained immunity refers to the ability of one antigenic stimulus to elicit more potent immune responses to a second, different antigen. Trained immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and is associated with epigenetic and metabolic changes. BCG is a potent tool as the first antigenic stimulus to elicit trained immunity to subsequent antigenic stimuli such as tumors, viral infection, or drug-resistant bacterial infections (Netea et al. Trained immunity: a program of innate immune memory in health and disease. Science 2016. PMID 27102489; and Arts et al. BCG vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity. Cell Host Microbe 2018. PMID 29324233).

[0211] BCG for solid and liquid tumors. BCG has a long history of therapeutic benefit as an immunotherapy for both solid and liquid tumors in humans (Hersh et al. BCG as adjuvant immunotherapy for neoplasia. Annu Rev Med 1977. PMID 324372). It has been used both systemically and intratumorally for malignancies that include melanoma, non-small cell lung cancer (NSCLC), and acute lymphoblastic leukemia (ALL). Recently there have been trials of BCG together with checkpoint inhibitors for forms of bladder cancer.

[0212] BCG for diabetes. BCG vaccination has recently been shown to have therapeutic benefits in glucose control for various forms of diabetes mellitus including Type 1 diabetes mellitus (Stienstra and Netea. Firing up glycolysis: BCG vaccination effects on Type 1 diabetes mellitus. Trends Endoc Metab 2018. PMID: 30327169). The effect is believed to be mediated by the trained immunity effects of BCG which have been shown to lead to epigenetic modifications which promote pro-inflammatory cytokine expression as well as the expression of metabolic enzymes such as those for glycolosis.

[0213] BCG-disA-OE and trained immunity. To investigate the ability of STING agonist overexpressing strains of BCG to stimulate trained immunity, the inventors tested the ability of BCG-WT versus BCG-disA-OE to elicit potentiation of second antigen stimulation in rested human monocytes following an exposure to the BCG strains six days prior. The first antigen was a BCG strain on day 0, and after six days of rest, the second antigen was the unrelated TLR-1/2 antigen PAM3CSK4. As may be seen in FIG. 20, upon receiving the second stimulus, the immune response tested (secretion of IL-1) was potentiated by both BCG-WT and BCG-disA-OE, but the degree of stimulation by BCG-disA-OE was statistically significantly greater than that of either no BCG first stimulus or BCG-WT as the first stimulus. This reveals that STING overexpressing BCG strains such as BCG-disA-OE are a more potent stimulators of trained immunity than BCG-WT.

[0214] In a related experiment, the inventors conduced the same BCG-first stimulation/6 day rest/TLR-1, 2 second antigen stimulation with PAM3CSK4 experiment with human monocytes. At the end of the experiment cellular DNA was collected and subjected to chromatin immunoprecipitation (ChIP) using an antibody for the H3K4 histone methylation mark. The H3K4 mark is a known transcriptional activation mark. Upon quantitative PCR amplification of the IL-6 promoter region of the immunoprecipitated DNA, the results showed that BCG-Pasteur-disA-OE and BCG-Tice-disA-OE were statistically significantly more potent in eliciting the H3K4 mark in the IL-6 promoter (IL-6 is a pro-inflammatory cytokine) than their respective BCG-WT strains. These results show that STING overexpressing BCG strains such as BCG-disA-OE are a more potent stimulators of epigenetic changes associated with trained immunity than BCG-WT.

BCG-Tice-disA-OE Expresses Much Higher Levels of the disA Gene than BCG-WT

[0215] As may be seen in FIG. 23, the relative expression of BCG-Tice-disA-OE clone 2 (which was selected for seed-lot preparation and storage) was 300:1 using the 2.sup.CT method of comparison. This indicates that disA is strongly overexpressed by being on a multicopy plasmid and driven by the M leprae hsp65 promoter in pSD5-hsp65-MT3692 plasmid. This strong overexpression leads to much higher levels of release of the STING agonist, c-di-AMP.

STING Agonist Overexpression BCG Strains Such as BCG-disA-OE Elicit Pro-Inflammatory Changes In Signaling Pathways and Cytokine Secretion Profiles in Multiple Model Systems.

[0216] The inventors tested STING agonist overexpressing strains such as BCG-disA-OE compared to BCG-WT in multiple model systems to evaluate its relative capacity to elicit proinflammatory cytokine changes. BCG-disA-OE was statistically significantly superior than BCG-WT in the majority of their tests. And when the comparisons were not statistically significant, BCG-disA-OE gave the stronger of the two responses. [0217] FIG. 25 also shows that the elevation of Type 1 IFN secretion in both BCG-disA-OE and BCG-WT is STING-dependent. [0218] In summary, BCG-disA-OE is a more potent stimulator of pro-inflammatory cytokine expression and proinflammatory pathway induction than BCG-WT [0219] The table below summarizes the data:

TABLE-US-00021 Mouse BMDM in vitro IRF3 qRT- BCG-disA-OE > BCG- FIG. 24 PCR WT Mouse BMDM, BMDC, IFN-, ELISA BCG-disA-OE > BCG- FIG. 26 J774 macrophage cell WT line in vitro Mouse BMDM; BMDC. IL-6 ELISA BCG-disA-OE > BCG- FIG. 27 J774 macrophage cell WT line in vitro Mouse BMDM, BMDC, TNF ELISA BCG-disA-OE > BCG- FIG. 28 J774 macrophage cell WT line in vitro Rat bladder cancer NBT-II TNF, IFN- ELISA BCG-disA-OE > BCG- FIG. 29 line in vitro WT Human bladder cancer IFN-, IFN-, ELISA BCG-disA-OE > BCG- FIG. 30 RT4 line in vitro TNF, IL-1 WT 5637, RT4, NBT-II IFN- qRT- BCG-disA-OE > BCG- FIG. 31 bladder cancer cell PCR WT lines In vitro Mouse lungs in vivo IFN-, IFN-, ELISA BCG-disA-OE > BCG- FIG. 32 (different time IL-6, TNF WT points) In vivo Mouse spleens in vivo IFN-; IFN-; ELISA BCG-disA-OE > BCG- FIG. 33 (4 weeks). In vivo IL-6; TNF WT
A Method to Produce an Antibiotic Gene Cassette-Free Recombinant BCG which Overexpresses a STING Agonist Biosynthetic Gene.

[0220] The disA-overexpressing plasmid pSD5-hsp65-MT3692 carries a Kan resistance gene cassette conferring resistance to the antibiotic kanamycin. The inventors disclose a method to generate an antibiotic gene cassette-free recombinant BCG which overexpresses a STING agonist biosynthetic gene.

[0221] The mycobacterial genetic operon panCD encodes for the biosynthetic gene panC (Pantoate-beta-alanine ligase gene) and panD (aspartate 1-decarboxylase gene). The gene products PanC and PanD are required for the biosynthesis of pantothenic acid also called vitamin B.sub.5 (a B vitamin). Pantothenic acid, a water-soluble vitamin, is an essential nutrient for mycobacteria such as BCG. Animals require pantothenic acid in order to synthesize coenzyme-A (CoA), as well as to synthesize and metabolize proteins, carbohydrates, and fats. The anion is called pantothenate. [0222] Genetic deletion of panCD in mycobacteria has been shown to yield mutant strains that can only grow in the presence of added pantothenate. As such they are auxotrophs for pantothenate. panCD mutants of Mycobacterium tuberculosis, have been shown to be highly attenuated in animal infection, being rapidly cleared, because of their inability to grow in mammalian tissues where pantothenate is not available to them. [0223] The inventors disclose a detailed method for generating an unmarked (no antibiotic gene cassettes) panCD deletion mutant of BCG. This mutant will only be able to grow in the presence of pantothenate and would not be expected to survive during infection or be an effective delivery vector for STING agonist expression. [0224] The inventors disclose a detailed method for generating a shuttle plasmid which harbors the mycobacterial panCD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA construct which overexpresses the disA gene and releases excess STING agonist, c-di-AMP). The shuttle plasmid is capable of replication in E. coli or in mycobacteria. It harbors an antibiotic cassette that can be conveniently removed by cleavage with a rare-cutting restriction enzyme and re-ligation. Alternatively, the shuttle plasmid may be generated by PCR amplification of the backbone of the plasmid excluding the antibiotic resistance cassette that generates unique restriction sites at the termini and ligating in a PCR product consisting of an amplified panCD operon with the same unique restriction sites at its termini. In either manner the antibiotic resistance gene-free shuttle plasmid (ligation product) may be electroporated into a BCG or E. coli auxotroph and selected for on pantothenate-free agar plates.

[0225] In the final manifestation of this disclosure, the inventors show a method to introduce the antibiotic-cassette-free plasmid harboring the mycobacterial panCD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA construct) into an unmarked BCG panCD mutant. The end result is a BCG strain that harbors no antibiotic resistance genes, and that strongly overexpresses a STING agonist biosynthetic gene(s). In a mammalian host or a human, such a BCG strain would be under strong selective pressure to retain the plasmid due to its requirement for panCD complementation from the plasmid. [0226] In another manifestation of the disclosure, the panCD cassette and the construct for the biosynthesis of STING agonists (such as the Phsp65::disA construct) could be introduced into a chromosomally integrating vector such as pMH94. Using similar methods the antibiotic cassette could be eliminated from pMH94. Introduction of this chromosomally integrating plasmid into an unmarked BCG panCD mutant would also yield a BCG strain that harbors no antibiotic resistance genes, and that strongly overexpresses a STING agonist biosynthetic gene(s). A disadvantage of this strategy is that the overexpression construct would be in single copy on the bacterial chromosome, rather than being in multicopy on a plasmid, and this could result in lower levels of STING agonist release.

BCG-Tice (ATCC 35743) is a Natural Pantothenate Auxotroph.

[0227] The inventors disclose that the Mycobacterium bovis BCG Tice strain (ATCC 35743) is a natural pantothenate auxotroph. This strain carries a 5 bp DNA insertion in its panC gene at base pairs 739-743. This insertion mutation changes leads to a frameshift mutation after the 246.sup.th amino acid of PanC (wild type PanC is 309 amino acids in length). As a result of the 5 bp insertion mutation, the mutant PanC polypeptide in the Mycobacterium bovis BCG Tice strain (ATCC 35743) is comprised of 246 amino acids of the wild type PanC sequence at its N-terminus followed by a 478 amino acid nonsense polypeptide at its C-terminus. This mutant PanC polypeptide is highly unlikely to retain any functional pantoate-beta-alanine ligase activity (the normal enzymatic function of PanC). Additionally, The PanD polypeptide in BCG Tice (ATCC 35743) is highly unlikely to be translated because the stop codon for the panC gene (which overlaps with the ATG for panD translation initiation in the wild type sequence) is out of frame. Ribosomal termination of PanC translation is coupled with ribosomal initiation of PanD translation in the wild type panCD operon. Since there is no ribosomal termination immediately upstream of the panD start codon, ribosomal initiation of translation of the panD gene is highly unlikely to occur.

[0228] The inventors disclose that this natural auxotrophy enables the more rapid construction of an antibiotic gene cassette-free recombinant BCG which overexpresses a STING agonist biosynthetic gene.

[0229] The inventors disclose a method for introducing an antibiotic-cassette-free plasmid harboring the mycobacterial panCD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA construct) directly into BCG-Tice (ATCC 35743).

[0230] Please note that pSD5-hsp60-MT3692 is the same as pSD5-hsp65-MT3692. The inventors had previously referred to this same plasmid as pSD5-hsp60-MT3692. However, the actual promoter in this strain is the promoter for the hsp65 gene of M. leprae. Thus, the inventors may refer to the plasmid pSD-hsp60-MT3692 as pSD5-hsp65-MT3692.

[0231] In some embodiments, the present invention relates to a pharmaceutical composition comprising an expression vector, expression cassette, or strain of the invention described herein and a pharmaceutically acceptable carrier.

[0232] In some embodiments, the present invention relates to methods and/or compositions for treating and/or preventing cancer comprising administration of an expression vector, expression cassette, strain or pharmaceutical composition described herein to a subject. In some embodiments, the cancer is bladder cancer (e.g., non-muscle invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor. Additional embodiments of the disclosure concern methods and/or compositions for treating and/or preventing a bladder cancer in which modulation of a Type 1 interferon (IFN) response is directly or indirectly related. In certain embodiments, individuals with a bladder cancer such as NMIBC are treated with a modulator of the Type 1 interferon response, and in specific embodiments an individual with bladder cancer is provided a modulator of expression Type 1 interferon expression, such as an inducer of its expression.

[0233] In certain embodiments, the level to which an inducer of Type 1 interferon expression increases Type 1 interferon expression may be any level so long as it provides amelioration of at least one symptom of bladder cancer, including non-muscle-invasive bladder cancer (NMIBC). The level of expression of Type 1 interferon may increase by at least 2, 3, 4, 5, 10, 25, 50, 100, 1000, or more fold expression compared to the level of expression in a standard, in at least some cases. An individual may monitor expression levels of Type 1 interferon using standard methods in the art, such as northern assays or quantitative PCR, for example.

[0234] An individual known to have bladder cancer, suspected of having bladder cancer, or at risk for having bladder cancer may be provided an effective amount of an inducer of Type 1 interferon expression, including a BCG strain of the present invention comprising an expression vector of the present invention. The expression vector expresses a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof; or a combination thereof. It is preferred that a BCG strain of the present invention comprising an expression vector of the present invention be administered into the bladder of the subject and that the expressed protein (s) enhance Type 1 interferon expression in the bladder. Those at risk for bladder cancer may be those individuals having one or more genetic factors, may be of advancing age, and/or may have a family history, for example.

[0235] In particular embodiments of the disclosure, an individual is given an agent for bladder cancer therapy in addition to the one or more inducers of Type 1 interferon of the present invention. Such additional therapy may include intravesical chemotherapies such as mitomycin C, cyclophosphamide, or a combination thereof, for example. When combination therapy is employed with one or more inducers of Type 1 interferon (such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof) the additional therapy may be given prior to, at the same time as, and/or subsequent to the inducer of Type 1 interferon.

[0236] In some embodiments, an expression vector, expression cassette, strain, pharmaceutical composition, and/or method of the invention described herein has increased safety, increased tolerability (e.g., decreased dysuria, urgency, or malaise), and/or decreased likelihood to cause infection in the bloodstream or disseminated bloodstream infection compared to non-recombinant BCG.

[0237] In some embodiments, the present invention relates to a method of treating and/or preventing cancer, comprising administering to a subject an expression vector, expression cassette, strain, and/or pharmaceutical composition of the invention described herein, wherein the administration results in an increased safety profile, increased tolerability (e.g., decreasing dysuria, urgency, or malaise), and/or decreased likelihood of infection in the bloodstream or disseminated bloodstream infection compared to non-recombinant BCG. In some embodiments, the cancer is, for example, bladder cancer (e.g., non-muscle-invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor. In some embodiments, the solid tumor is, for example, a sarcoma, carcinoma, or lymphoma.

[0238] In some embodiments, the present invention relates to a method of increasing the safety, increasing the tolerability (e.g., decreasing dysuria, urgency, or malaise), and/or decreasing the likelihood to cause infection in the bloodstream or disseminated bloodstream infection compared to non-recombinant BCG, comprising administering an expression vector, expression cassette, strain, and/or pharmaceutical compositions of the invention described herein to a subject.

Pharmaceutical Preparations

[0239] Pharmaceutical compositions of the present invention comprise an effective amount of one or more inducers of expression of Type 1 interferon such as such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase pharmaceutical or pharmacologically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that comprises at least one inducer of expression of Type 1 interferon or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21.sup.st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0240] As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0241] The inducer of expression of Type 1 interferon (such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof) may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. In some embodiments, the present invention (e.g., expression vectors, strains, or pharmaceutical compositions) can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intravesically (e.g., administered directly into the bladder, e.g., by injection, or by intravesical instillation), intratumorally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, and Francica et al. TNF and radio-resistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide STING agonists in non-immunogenic tumors. Cancer Immunol Res. 2018 Feb. 22. PMID: 29472271, incorporated herein by reference).

[0242] Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

[0243] Further in accordance with the present disclosure, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0244] In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

[0245] In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0246] In further embodiments, the present invention includes the use of pharmaceutical lipid vehicle compositions that include inducer of expression of Type 1 interferon, one or more lipids, and an aqueous solvent. As used herein, the term lipid includes any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term lipid is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

[0247] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the inducer of inducer of expression of Type 1 interferon of the present invention may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0248] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0249] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0250] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

A. Alimentary Compositions and Formulations

[0251] In one embodiment of the present disclosure, the inducers of expression of inducer of expression of Type 1 interferon of the present invention are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0252] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz E, Jacob J S, Jong Y S, Carino G P, Chickering D E, Chaturvedi P, Santos C A, Vijayaraghavan K, Montgomery S, Bassett M, Morrell C. Biologically erodable microspheres as potential oral drug delivery systems. Nature. 1997;386:410-4. PMID: 9121559; Hwang M J, Ni X, Waldman M, Ewig C S, Hagler A T. Derivation of class II force fields. VI. Carbohydrate compounds and anomeric effects. Biopolymers. 1998;45:435-68. PMID: 9538697; Hwang J S, Chae S Y, Lee M K, Bae Y H. Synthesis of sulfonylurea conjugated copolymer via PEO spacer and its in vitro short-term bioactivity in insulin secretion from islets of Langerhans. Biomaterials. 1998;19:1189-95. PMID: 9720902; Hwang S J, Park H, Park K. Gastric retentive drug-delivery systems. Crit Rev Ther Drug Carrier Syst. 1998;15:243-84. PMID: 9699081; U.S. Pat. Nos. 5,641,515; 5,580,579; and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0253] For oral administration the compositions of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

[0254] Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

B. Parenteral Compositions and Formulations

[0255] In further embodiments, inducer of expression of Type 1 interferon of the present invention may be administered via a parenteral route. As used herein, the term parenteral includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally. See, e.g., U.S. Pat. Nos. 6,7537,514; 6,613,308; 5,466,468; 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

[0256] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.

[0257] Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0258] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see, for example, Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.

[0259] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

[0260] In other preferred embodiments of the invention, the active compound inducer of expression of Type 1 interferon of the present invention may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation. Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a patch. For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

[0261] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga M, Serizawa Y, Azechi Y, Ochiai A, Kosaka Y, Igarashi R, Mizushima Y. Microparticle resins as a potential nasal drug delivery system for insulin. J Control Release. 1998; 52:81-7. PMID: 9685938) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety). The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.

Kits of the Disclosure

[0262] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an inducer of expression of Type 1 interferon of the present invention (such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof) may be comprised in a kit.

[0263] The kits may comprise a suitably aliquoted inducer of expression of Type 1 interferon of the present invention and, in some cases, one or more additional agents. The component(s) of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the inducer of expression of Type 1 interferon of the present invention and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0264] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The inducer of expression of Type 1 interferon of the present invention composition(s) may be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.

[0265] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0266] The Examples above have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the Examples above are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The Examples above are offered by way of illustration and not by way of limitation.

[0267] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0268] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0269] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.