CA3167223A1 - Recombinant therapeutic interventions for cancer - Google Patents

Abstract

Described are methods of suppressing the expression of myeloid-derived suppressor cells (MDSCs), M2 macrophages, and Treg cells in a tumor and inducing the expression of macrophages, dendritic cells (DCs), and T effector cells in a tumor in a subject. A pharmaceutical composition comprising a strain of Mycobacteria including an expression vector of the present invention is administered to a subject.

Description

2 RECOMBINANT THERAPEUTIC INTERVENTIONS FOR CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application claims the benefit of priority under 35 U.S.C. 119 (c) of U.S. Patent Application No. 16/790,161 filed on February 13, 2020, which is a continuation-in-part (CIP) of 16/638,943 filed on February 13,2020, that is a 35 U.S.C. 371 U.S. national entry of International Application PCT/US2019/022341, having an international filing date of March 14, 2019, which claims the benefit of U.S. Provisional Application No. 62/658,661, filed April 17, 2018, the content of each of the aforementioned applications is herein incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, named JHU4280_2W0 Sequence Listing.txt, was created on February 11, 2021, and is 157 kb. The file can be assessed using Microsoft Word on a computer that uses Windows OS.
STATEMENT OF GOVERNMENTAL INTEREST

[0003] This invention was made with government support under grant nos. AI036973, AI037856, awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION

[0004] Urothelial cancer of the bladder is the most common type of bladder cancer (BC) in North America, South America, Europe and Asia. Non-Muscle Invasive Bladder Cancer (NMIBC) is associated with a high recurrence rate, frequent intravesical treatments, risk of progression to advanced stages and the highest lifetime treatment among all cancers.
Intravesical BCG (bacillus Calmette Guerin) instillation has been the standard of care treatment for NMIBC for 30 years. It is effective in 60-70% patients. BCG has shown to be a very effective vehicle for delivery of antigens. Many studies corroborating an underlying immune response skewed towards a Type I
interferon and Th I induced mediated immune response show promise. Efforts to generate recombinant BCG (rBCG) strains for NMIBC have focused on developing strains that augment these anti-tumor immune responses. To date such efforts have not yielded demonstrable improvement over traditional BCG.

SUMMARY OF THE INVENTION

[0005] One embodiment of the present invention provides a vector including a nucleic acid sequence expressing a protein or functional part thereof that makes a STING
agonist including c-di-AMP (also known as 3'-5' c-di-AMP); c-di-GMP (also known as 3"-5' c-di-GMP); 3'-3'cGAMP (also known as 3'-5', 3'-5'cGAMP, the product of the Vibrio cholerae DncV protein);
2' -3'cGAMP (also known as 2' -5' , 3'-5' cGAMP, the product of the human cGAS
protein) and a combination thereof, as examples. Some vectors of the present invention include a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Ry1354c 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. Each of these nucleic acid sequences express proteins that make one or more of the STING agonist as described in the definition section of the specification. Some vectors of the present invention include in addition to one or more of the sequences listed above a fifth nucleic acid sequence encoding a PanC protein and a PanD protein or functional part thereof.
Vectors including a nucleic acid sequence encoding a PanC protein and a PanD
protein or functional part thereof are typically free of an antibiotic resistance gene.
Suitable vectors used in the present invention may include vectors that replicate episomally in multiple copies, or vectors that integrate into a bacterial chromosome in single copy or are otherwise present in the bacterial cell. A vector of the present invention may stably integrate into a bacterial genome or it may stably replicate as an episomal plasmid. Suitable third nucleic acid sequences include those that overexpress the cyclase domains of the cyclic GMP-AMP synthase (cGAS) protein.
Other suitable third nucleic acid sequence may express a cyclic GMP-AMP synthase (cGAS) protein haying a regulatory DNA recognition capability that is non-functional. Vectors of the present invention may also include nucleic acid sequences that encode sequences or proteins that knock out the expression of PDE genes of a strain of Mycobacteria used in the present invention.

[0006] Another embodiment of the present invention provides a strain of Mycobacleria including any one of the vectors of the present invention including a vector comprising a protein or functional part thereof that makes a STING agonist. As mentioned above, examples of STING
agonist include c-di-AMP (also known as 3'-5' c-di-AMP); c-di-GMP (also known as 3'-5' e-di-GMP); 3'-3'cGAMP (also known as 3'-5', 3'-5'cGAMP, the product of the Vibrio cholerae DncV
protein); 2'-3'cGAMP (also known as 2'-5', 3'-5' cGAMP, the product of the human cGAS
protein) and a combination thereof, as examples. Examples of suitable nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Ry1354c 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 (dis A) protein, or a functional part thereof and a combination thereof. Examples of suitable strains of Mycobacterium used in the present invention include Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof, for example. Another strain used in the present invention is Mycobacterium bacillus Calmette Guerin (BCG). A strain of Mycobacteria used in the present invention may be a panthothenate auxotroph of BCG lacking its pan CD genetic operon. pan CD
auxotoph strains lack genomic sequences able to encode functional PanC and/or PanD protein. In some embodiments, strains of Mycobacteria that are pantothenate auxotrophs comprise vectors of the present invention including apanCD nucleic acid encoding the PanC and PanD proteins or functional parts thereof.
Vectors of the present invention that include panCD nucleic acid sequences are preferably free of antibiotic resistant genes or nucleic acid sequences that encode functional proteins providing antibiotic resistance. Mycobacteria that are pantothenate auxotrophs of the present invention are preferably free of a genomic antibiotic resistant gene or unable to encode functional proteins that provide antibiotic resistance.

[0007] Another embodiment of the present invention provides a pharmaceutical composition, including any one of the strains of Mycobacteria of the present invention, and a pharmaceutically acceptable carrier.

[0008] Another embodiment of the present invention provides a method of eliciting a type 1 interferon response, enhancing the expression of pro-inflammatory cytokine, and/or eliciting trained immunity in a subject including the steps of: administering a pharmaceutical composition including anyone of the strains of the present invention into a subject; and eliciting a type 1 interferon response, enhancing the expression of pro-inflammatory cytokine, and/or eliciting trained immunity in the subject. In one aspect, the pharmaceutical composition is administered into the bladder of the subject by a catheter.
100091 Another embodiment provides a method of using a strain of Mycobacteria of the present invention to treat or prevent cancer in a subject. The method includes the steps of:
administering a pharmaceutical composition including a strain of Mycobucteriu including 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. The present invention may be used to treat or prevent cancers including epithelial cancers, breast cancer, non-muscle invasive bladder cancer, as examples. In some aspects, the cancer is a BCG-unresponsive non-muscle invasive bladder cancer (BCG-unresponsive NMIBC) and the pharmaceutical composition is administered by intravesical instillation. In some aspects, the cancer is a BCG-ntifive non-muscle invasive bladder cancer (BC G -n alve NM IBC) and the pharmaceutical composition is administered by i ntrav es i c al instillation. In other aspects, the 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 to a luminal surface of the epithelial cancer. In some aspetcs, the cancer is selected from a solid tumor or a liquid tumor and the pharmaceutical composition is administered by intratumoral injection and/or by systemic infusion. The methods of the present invention may include the step of administering a checkpoint inhibitor, such as an anti-PD1 antibody, an anti-PDL1 antibody, or a combination thereof, as example. In another aspect, the cancer is bladder cancer and the pharmaceutical composition is administered via a catheter.
100101 One embodiment of the present invention provides an expression vector including a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA
integrity scanning (disA) protein which functions as a diadenylate cyclase, or a functional part thereof, or a combination thereof Some expression vectors of the present invention include a first nucleic acid sequence that overexpresses the cyclase domains of the Rv1354c protein when compared to the expression of a native Rv1354c protein as a reference. Some expression vectors of the present invention include a second nucleic acid sequence that overexpresses the cyclic GMP-AMP synthase (DncV) protein, when compared to the expression of a native DncV protein. Some expression vectors of the present invention include a third nucleic acid sequence that vet-expresses the cyclasc domains of the cyclic GMP-AMP synthase (cGAS) protein when compared to the expression of a native cGAS protein.
Suitable Rv1354 proteins used in the present invention include a Mycobacterium tuberculosis Rv1354 protein. Suitable DncV proteins used in the present invention include a Vibrio cholera DncV protein. Suitable cGAS proteins used in the present invention include a Homo sapiens cGAS
protein. Suitable DisA proteins used in the present invention include a Mycobacterium tuberculosis disA protein.
[0011] Another embodiment of the present invention provides a strain of BCG including a cdnP gene, an Rv1354c gene, an Rv1357c gene, or a combination thereof, wherein the cdnP gene is unable to express a functional cyclic di-nucleotide phosphodiesterase (CdnP) protein, the Rv1354e gene is unable to express a functional Rv1345c protein, and/or the Rv1357c gene is unable to express a functional Rv1357 protein. Some BCG strains of the present invention may have an Rv1354c gene that includes a non-functional EAL domain. The BCG
strains of the present invention may include any of the expression vectors of the present invention.
[0012] Another embodiment of the present invention provides a method of treating or preventing bladder cancer including the steps of: administering a pharmaceutical composition including a strain of BCG including an expression vector of the present invention into the bladder of a subject; and treating or preventing bladder cancer in the subject when compared to a reference subject who was not administered the pharmaceutical composition. The pharmaceutical composition may be administered by any suitable means including by a catheter.
[0013] Another embodiment of the present invention provides a method of eliciting a type 1 interferon response in a subject including the steps of: administering a pharmaceutical composition including a strain of BCG including an expression vector of the present invention into the subject such as the subject's bladder; and enhancing a type 1 interferon response in the subject compared to a reference subject not administered the pharmaceutical composition.
[0014] Another embodiment of the present invention provides a method of treating or preventing cancer in a subject including the steps of: administering a pharmaceutical composition comprising a strain of BCG including an expression vector of the present invention into a tumor of a subject having cancer; and treating or preventing cancer in the subject when compared to a reference subject not administered the pharmaceutical composition. The pharmaceutical composition may be administered by any suitable means including injection into the tumor.
Cancers that may be treated or prevented by this method include, but are not limited to, breast cancer, and/or non-muscle invasive bladder cancer.
[0015] Examples of Mycobacteria used in the present invention include Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis Bacillus Calmette Guerin (referred to a BCG), Mycobacterium smegmatis, Mycobacterium avium complex, and other non-tuberculous mycobacteria (NTM). Examples of BCG strains used in the present invention including those that overexpress STING agonists, include BCG Pasteur, BCG-Pasteur-Aeras, BCG Tice (also known as BCG Chicago), BCG-Connaught (also known as BCG Toronto), BCG Danish, BCG-Prague (also known as BCG Czechoslovakian), BCG Russia (also known as BCG Moscow), BCG Moreau (also known as BCG Brazil), BCG Japan (also known as BCG Tokyo), BCG Sweden (also known as BCG Gothenburg), BCG Birkhaug, BCG Glaxo, BCG Frappier (also known as BCG
Montreal), BCG Phipps, or other available BCG strains.
[0016] Another embodiment of the present invention provides a method of treating diabetes including the steps of: administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject having diabetes; and treating or preventing diabetes in the subject by providing trained immunity. Trained immunity refers to the ability of one antigenic stimulus to elicit more potent immune responses to a second, different antigenic stimulus introduced at a later time. Trained immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and is associated with epigenetic and metabolic changes. The method results in the upregulation of glycolysis mediated by the trained immunity. The aforementioned up-regulation of glyeolysis is beneficial in preventing and treating type 1 and type 2 diabetes mellitus.
[0017] Another embodiment of the present invention provide a method of stimulating trained immunity in a subject including the steps of: administering a pharmaceutical composition including a strain of Mycohacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject; and stimulating trained immunity in the subject.
Upregulating glycolysis in the subject and/or stimulating episomal changes in hi stone methylation in the subject mediate trained immunity in the subject.
[0018] Another embodiment of the present invention provides a method of treating or preventing a viral infection in a subject including the steps of:
administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject; and treating or preventing the viral infection in the subject. Stimulating trained immunity in the subject treats or prevents the viral infection in the subject. Upregulating glycolysis in the subject and/or stimulating episomal changes in histone methylation in the subject mediate trained immunity in the subject.
[0019] Another embodiment of the present invention provides a method of treating or preventing a bacterial infection, or a drug-resistant bacterial infection in a subject including the steps of: administering a pharmaceutical composition including a strain of /14-ycobacteria including a vector expressing a protein or a functional part thereof that makes a STING
agonist to a subject;
and treating or preventing the bacterial infection or the drug-resistant bacterial infection in the subject. Stimulating trained immunity in the subject treats or prevents the bacterial infection in the subject. Upregulating glycolysis in the subject and/or stimulating episomal changes in histone methylation in the subject mediate trained immunity in the subject. The methods of the present invention may use one or more of the vectors of the present invention or one or more strain of bacteria including a vector of the present invention.
[0020] Another embodiment of the present invention provides a method of suppressing the expression of myeloid-derived suppressor cells (MDSCs), M2 macrophages, and Treg cells in a tumor and inducing the expression of macrophages, dendritic cells (DCs), and T
effector cells in a tumor. The method includes the steps of administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein that makes a STING agonist or a functional part thereof to a subject having a tumor; suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor; and inducing the expression of macrophages, DCs, and T effector cells in the tumor. An example of Ml macrophages having induced expression in a tumor includes Ml macrophages. An example of T effector cells having induced expression in a tumor includes CD4+ T cells and CDS+ T cells. Suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor of subjects administered a Mycobacteria including a vector of the present invention is observed when compared to the expression of MDSCs, M2 macrophages, and Treg cells in a tumor of a referenced subject not administered a pharmaceutical composition including the strain of Mycobacteria. Inducing the expression of macrophages, DCs, and T effector cells in a tumor is observed when compared to the expression of macrophages, DCs, and T effector cells in a tumor of a referenced subject not administered a pharmaceutical composition comprising the strain of Mycobacteria. Examples of suitable STING
agonist include 3'-5' c-di-AMP (also known as c-di-AMP); 3'-5' c-di-GMP (also known as c-diGMP); 3'-3' cGAMP; 2'-3'eGAMP and a combination thereof. A suitable vector of the present invention may include a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Ry1354c 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. The tumor may be a epithelial cancer, a breast cancer, or a non-muscle invasive bladder cancer, and melanoma as examples. In some aspects, the tumor may be a non-muscle invasive bladder cancer such as a BCG-unresponsive non-muscle invasive bladder cancer (BCG-unresponsive NMIBC) and the pharmaceutical composition can be administered by intravesical instillation. In other aspects, the tumor may be a non-muscle invasive bladder cancer such as a BCG-naive non-muscle invasive bladder cancer (BCG-nalve NMIBC) and the pharmaceutical composition can be administered by intravesical instillation. In other aspects, the tumor may be an epithelial cancer 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 can be administered to a luminal surface of the epithelial cancer. In other aspects, the tumor is a solid tumor and the pharmaceutical composition is administered by intratumoral, intravenous, intradermal, transdermal, intravesical topical, intramuscular or subcutaneous injection. In other aspects, the tumor is a liquid tumor and the pharmaceutical composition is administered by intravenous, intradermal, transdermal, intravesical topical, intramuscular or subcutaneous injection. Methods of the present invention may further comprise the step of administering a checkpoint inhibitor. Suitable checkpoint inhibitors that may be used in the present invention include ipilimumab (anti-CTLA-4 antibody), nivolumab (anti-PD-1 antibody), pembrolizumab (anti-PD-1 antibody), cemiplimab (anti-PD-1 antibody), atezolizumab (anti-PD-L1 antibody), avclumab (anti-PD-Li antibody), durvalumab (anti-PD-L1 antibody) and a combination thereof BRIEF DESCRIPTION OF THE DRAWINGS
[0021] This application is continuation- in- part (CIP) of 16/638,943, all the figures from US
serial number 16/638,943 are herein incorporated by reference in their entirety.
[0022] FIGs 1A-1B Mycobacteria overexpressing disA from the pSD5B
Phsp6o::disA plasmid construct release large amounts of c-di-AMP into the macrophage cytosol and transcribe high levels of disA mRNA. FIG. 1A. J774 macrophages infected with M.tb harboring the pSD5B
Phsp60::disA plasmid or wild type M.tb (CDC1551) at all 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 RJ, Cheung LS, Pokkali S, Guo H, Lee JH, Bishai WR. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis. Nat Med. 2015;21:401-6. PMID: 25730264.) FIG. 1B. BCG-Pasteur harboring the pSD5B
Phsp6o::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.
[0023] FIG. 2. BCG overexpressing disA augments pro-inflammatory eytokines. Gene expression profiling (qPCR) of pro-inflammatory cytokines and IFNA3 in mouse BMDMs challenged with wild-type and disA overexpression strains of BCG-Pasteur.
[0024] FIG. 3. BCG overexpressing disA augments IRF3 signaling.
Effect of disA
overexpression on activation of IRE 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.

9 [0025] FIG s 4A-4C. Increased pro-inflammatory cytokines in response to disA
overexpression. FIG. 4A shows differential expression of TNF-a. FIG. 4B shows differential expression of IL-6. FIG. 4C shows differential expression of IL-113.Mouse BMDMs were challenged with wild-type and disA overexpression strains of BCG-Pasteur.
Culture supernatants were assayed by ELISA for different cytokines.
[0026] 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.
[0027] FIG. 6. Schematic workflow of testing relative therapeutic efficacy of wild-type and BCG-disA-OE strains.
[0028] FIG. 7. Tumor involvement index of tumor-bearing rats untreated or treated with WT
BCG or rBCG overexpressing disA (rBCG = BCG-Pasteur-disA-0E; wtBCG = BCG-Pasteur).
[0029] 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.
[0030] FIG. 9. Gene expression profiling of bladders from MNU
tumor bearing rats untreated or treated with WT or rBCG overexpressing disA.
[0031] 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.
[0032] FIG. 11. 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.
[0033] FIG. 12. M. tuberculosis harboring the pSD5B Phsp60::disA
plasmid (Mtb-disA-OE or Mtb-OE) is significantly attenuated for virulence in mice compared to wild type M.tb (Mtb-CDC1551). 6-7-week-old female BALB/c mice (n = 10 per group) were infected as described above with ¨3.5 logio 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 Mtb-disA-OE
(Mtb-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 RJ, Cheung LS, Pokkali S, Guo H, Lee JH, and Bishai WR. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis. Nat Med.
2015; 21: 401-6.
PMID: 25730264.) [0034] FIGs 13A-13B. Other BCG strains are also active: BCG Tice strain overexpressing disA also shows induction of proinflammatory cytokincs 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 EL1SA. FIG. 13A
shows Bdifferential expression pattern of TNF-a. FIG. 13B shows differential expression of IL-6.
Mouse BMDM
were challenged with the two different strains of BCG. The BCG-Tice strain was from the commerci ally avail able Onco-Tice product.
[0035] FIG. 14. 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 1EN- 13 levels using EL1SA.

[0036] FIG. 15 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 MN U 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, Ti, 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 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 (Figure 15) represent the combined results of Experiment 1 plus Experiment 2.
The qPCR data shown in Figure 8 and Figure 9 were obtained using bladder tissue at necropsy from the end of Experiment 1.
[0037] FIG. 16 shows that BCG-disA-OE reduces Tregs (CD4+CD25+Foxp3 ) in murine syngeneic bladder cancer tumors. Mice were implanted on the flank with 5 x 106 BBN975 murine bladder cancer tumor cells. When the tumors were 1.5 cm in diameter, mice received 3 intratum oral 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 C134 Tregs, reduced tumor CD8 Tregs, and reduced spleen CD4+ Tregs.
[0038] FIGs 17A-17B show that BCG-disA-OE is safer than BCG-WT in two mouse models.
FIG. 17A shows that groups of BALB/e mice (immunocompetent) were exposed to 1 x 103 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. FIG. 17B shows that groups of SCID
mice (immunosuppressed) were exposed to 1 x 102 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-0E-infected mice had a statistically significantly longer survival time than BCG-WT-infected mice.
[0039] FIG. 18 shows that BCG-disA-OE elicits statistically significantly higher levels of "Trained Immunity immunological and epigenetic marks" in CD14+ 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, CD lzkhuman monocytes were prepared from LeukoPaks collected by apheresis. On day 0 they were infected with either BCG-WT or BCG-disA -OE at a MOT 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 13 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.
[0040] FIG. 19 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 region of cytokines and other immune mediators. The experiment shown in Figure 19 was performed in the same set of cells and exactly the same way as that described in Figure 18 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 (ChM) using an antibody specific for the H3K4-me3 histone methylati on 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-di.sA-OE and BCG-Tice-disA-OE led to significantly greater levels of H3K4 histonc trimethylation in thc 1L6 and TNF
promoter regions than did their corresponding BCG-WT strains following challenge with the second stimulus, PAM3C SK4.
[0041] FIG. 20 shows the successful construction of BCG-Tice-disA-0E. The inventors' previous work had utilized BCG-Pasteur to construct BCG-Pasteur-disA-0E. 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 purchased 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.
[0042] FIG. 21 shows that clone 2 ofBCG-Tice-disA -OE from the transformation experiment shown in Figure 20 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-AAcT 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-0E" in all subsequent work.
[0043] FIG. 22 shows potent, statistically significantly enhanced 1RF3 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 3h. 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-'2T method. Student's t test followed by Welch correction (***P<0 001 ; **P<0.01).
[0044] FIG. 23 shows that STING is required for enhanced type I
IFN (TENT) 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-p was performed in culture supernatants as per the manufacturer's instruction.
Data points represent the mean of three independent biological experiments standard error mean (S.E.M.). Student's t test followed by Welch correction (**P<0.01).
[0045] FIGs 24A-24C shows that interferon-3 is induced murine BMDMs, BMDCs and J774.1 macrophages in upon exposure to disA overexpressing BCG strains and that the TENT
response is statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Ti ce-disA -OE
than for the corresponding BCG-WT strains. Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3h using different strains of BCG (MOI: 20). Non-internalized bacilli were washed using warm DPBS and cell were incubated for another 24 hours. IFN-P levels were quantified in culture supernatants using ELISA as per manufacturer's instruction. Data points represent three independent biological experiments+
standard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001;
**P<0.01; *P<0.05). FIG. 24A shows interferon-13 levels in murine BMDMs. FIG.
24B shows interferon-I3 levels in murine BMDCs. FIG. 24C shows intcrferon-P levels in murine J774.1 macrophages.
[0046] FIGs 25A-25C show 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. 1L-6 levels were quantified in culture supernatants using EL1SA as per manufacturer's instruction. Data points represent three independent biological experiments standard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001;
**13<0.01; *P<0.05). FIG. 25A shows IL-6 levels in murine BMDMs. FIG. 25B
shows IL-6 levels in murine BMDCs. FIG. 25C shows IL-6 levels in murine J774.1 macrophages.
[0047] FIGs 26A-26C 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 3h using different strains of BCG (MOT:
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 experiments standard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001;
**P<0.01; *P<0.05).
FIG. 26A shows TNF levels in murine BMDMs. FIG. 26B shows TNF levels in murine BMDCs.
FIG. 26C shows TNF levels in murine J774.1 macrophages.
[0048] FIGs 27A-27B shows that TNF and IFN-y are induced 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-1I cells were infected with wild-type and recombinant strains of BCG for 3h. 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-y. Data points represent three independent biological experiments standard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.0001;
**P<0.001;
*P<0.05). FIG. 27A shows TNF levels in NBT-II cells. FIG. 27B shows IFN-y levels in NBT-II
cells.

[0049] FIGs 28A-28D shows that of IFN-13, IFN-y, TNF and IL-113 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 thc corresponding BCG-WT strains. RT4 cells were infected with wild-type and recombinant strains of BCG for 3h. 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 experiments+ standard error mean (S.E.M.). Data analysis was performed using unpaired t-test (***P<0.001; **P<0.01; *P<0.05). FIG. 28A shows IFN-13 levels in RT4 cells. FIG. 28B shows IFN-y levels in RT4 cells. FIG. 28C shows TNF levels in RT4 cells. FIG.
28D shows IL-1[3 levels in RT4 cells.
[0050] FIG. 29 shows that BCG-disA-OE stimulates increased IFN-13 levels in multiple bladder cancer cell lines to a greater degree than BCG-WT. The drawing shows the levels of -ITN-13 mRNA (relative expression by the 2-A 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.
[0051] FIGs 30A-30D shows the cytokine responses for IFN-13, IFN-y, 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 Figure 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 group S.E.M.).
Data analysis was performed using paired t-test (***P<0.001; **P<0.01;
*P<0.05). FIG_ 30A
shows IFN-[3 levels in BCG-WT and BCG-c/isA-0E-infected mouse lungs. FIG. 30B
shows IFN-y levels in BCG-WT and BCG-disA-0E-infected mouse lungs. FIG. 30C shows IL-6 levels in BCG-WT and BCG-disA-0E-infected mouse lungs. FIG. 30D shows TNF levels in BCG-WT and BCG-disA -OE-infected mouse lungs.

[0052] FIG s 31A-31D shows the cytokine responses for IFN-y, IL-6, and TNF in BCG-WT and BCG-disA-0E-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-Ticc-disA-OE than for the corresponding BCG-WT strains. BALB/c mice were infected by the aerosol route as described in Figure 17. 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 group S.E.M.). Data analysis was performed using paired t-test (***P<0.001; **P<0.01; *P<0.05). FIG. 31A shows IFN43 levels in BCG-WT and BCG-disA-0E-infected mouse spleens. FIG. 31B shows IFNI, levels in BCG-WT
and BCG-disA-0E-infected mouse spleens. FIG. 31C shows IL-6 levels in BCG-WT
and BCG-disA-0E-infected mouse spleens. FIG. 31D shows TNF levels in BCG-WT and BCG-disA-0E-infected mouse spleens.
[0053] FIG. 32 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
ID NO:30) with the panCD operon to generate "pSD5.hsp65-disA.panCD--No Kan" (SEQ ID
NO:31).
[0054] FIG. 33 shows the molecular structure of the pJV53, the recombineering plasmid which is SEQ ID NO:32 [0055] FIG. 34 shows the molecular structure of the pUC-Hyg, a plasmid with dif sites flanking a Hyg cassette which is SEQ TD NO:35. pUC-Hyg is used to generate the plasmid "pUC-Hyg-panCD-KO" (SEQ ID NO:36).
[0056] FIG. 35 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 5'UTR
on one flank of the Hyg cassette, and cloning 500 bp of the panCD 3' UTR the other flank.
[0057] FIG. 36 shows the molecular structure of the plasmid "pSD5.hsp65-disA.Kan" which is SEQ ID NO:30.
[0058] FIG. 37 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 Figure 32.
[0059] FIG. 38 shows the number of positive specimens.

[0060] FIGs 39A-39C shows confirmation of Mtb-disA overexpression phenotype of BCG-disA -OE and induction of TRF signaling. Fig. 39A shows colony PCR using Kanamycin gene specific primer confirms the presence of recombinant plasmid pSD5-hsp60-MT3692 in the BCG-disA-OE (Tice) clones selected against Kanamycin (25 mg/mL). Fig. 39B shows real time PCR
showing differential disA expression in different clones of BCG Tice BCG-disA-OE Tice.
Transcript levels were measured in total RNA isolated from the late log phase cultures using log phase culture. M tuberculosis sigA (Rv2703) was used as a reference gene, and relative expression was calculated by 2AACT method. Fig. 39C shows measurement of IRF activation BY quantification of IRF induction based on ISRE (RLU, relative light units) in 24-h-post infection (MOI = 1:20) culture supernatants of RAW-Lucia ISG cells. Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0061] FIGs 40A-40C show BCG strains overexpressing c-di-AMP as strong inducers of type I interferon in STING-dependent manner. Fig. 40A shows quantitative measurement of IFN-p in culture supernatants of wild-type C57BL/6-derived BMDMs and STING-KO BMDM
(C57BL/6J-Tmem173gt/J) 24 h after BCG-disA -OE (Tice) infection. Fig. 40B shows quantitative measurement of IFN-p in culture supernatants of wild-type C57BL/6-derived BMDMs, BMDCs, J774.1 macrophages and human monocyte-derived macrophages (RMDMs) 24 h post-infection.
Fig. 40C shows quantitative measurement of IFNI!, in culture supernatants of wild-type C57BL/6-derived BMDMs, BMDCs, J774.1 macrophages and human monocyte-derived macrophages (HMDMs) 24 h post-infection. Macrophage to BCG infection ratio = 1:20. Data represent mean SEM (n = 3 replicates). Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****1)<
0.0001 [0062] FIGs 41A-41D show BCG strains overexpressing c-di-AMP are strong inducers of proinflammatory cytokines, TNF-a, and IL-6. (A-B) Quantitative measurement of TNF-ct in culture supernatants of wild-type C57BL/6-derived BMDMs, BMDCs, J774.1 macrophages and human monocyte-derived macrophages (HMDMs). Fig. 41A shows measurements 24 h post-infection with BCG-disA-OE (Tice). Fig. 41B shows measurements 24 h post-infection with BCG-disA-OE (Pasteur). (C-D) Quantitative measurement of IL-6 in culture supernatants of wild-type C57BL/6-derived BMDMs, BMDCs, J774.1 macrophages and human monocyte-derived macrophages (HMDMs). Fig. 41C shows measurements 24 h post-infection with BCG-disA-OE

(Tice). Fig. 41D shows measurements 24 h post-infection with BCG-disA-OE
(Pasteur).
Macrophage to BCG infection ratio = 1:20. Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0063] FIG. 42 shows BCG overexpressing c-di-AMP strongly induces proinflammatory cytokine INF-a in STING-dependent manner. Quantitative measurement of TNF-a in culture supernatants of wild-type C57BL/6-derived BMDMs and STING-KO BMDM (C57BL/6J-Tmem173gt/J) 24 h after BCG-disA-OE (Tice) infection. Macrophage to BCG
infection ratio 1:20. Data represent mean SEM (n = 3 replicates). Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0064] FIGs 43A-43E show BCG-disA-OE induces significantly higher Thl cytokines and chemokines as compared to WT BCG. Fig. 43A shows relative gene expression analyses of different cytokines and chemokines in IFN-y activated macrophages at 6 h post-infection by wild-type BCG (Tice) and BCG-disA-OE (Tice) strains. (B-D) Relative gene expression analyses of IL-6, 1L-12 and MCP-1 1FN-y activated macrophages at 6 h post-infection by wild-type BCG
(Pasteur) and BCG-disA -OE (Pasteur) strains. ft-actin was used as a reference gene, and relative expression was calculated by 2AACT method. Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed). Fig. 43B shows IL-6 relative gene expression.
Fig. 43C shows IL-12 relative gene expression. Fig. 43D shows MCP-1 relative gene expression. Fig.
43E shows quantitative measurement of MCP-1 in culture supernatants of wild-type C57BL/6-derived BMDMs 24 h after BCG-disA-OE (Tice) infection. Macrophage to BCG infection ratio = 1:20.
Data represent mean SEM (n = 3 replicates). Student's t-test (two-tailed) *P<0.05, **P<0.01, ****P< 0.0001 [0065] FIGs 44A-44C show differential apoptotie induction in murine BMDMs and J774.1 macrophage after infection with different BCG strains. (A-B) Murine BMDMs and J774.1 macrophages were challenged with WT or BCG-disA-OE strains of BCG at a MOI of 1:10 for 24 h and apoptotic cell death was accessed by Annexin and PI staining.
Representative data from individual infection assay. Fig. 44A shows measurements in murine BMDMs. Fig.
44B shows measurements in J774.1 macrophages. Fig. 44C shows a bar diagram showing quantifications of late apoptotic cell death following infection. Data represent mean SEM (n =
3 replicates).
Student's t-test (two-tailed) *P<0.05 [0066] FIGs 45A-45C show internalization and differential toxicity of WT and BCG-disA-OE
strains in human urothelial carcinoma cells. (A-C) Cell viability of RT4 (Human bladder cancer cell line representing grade I carcinoma), 5637 (Human bladder cancer cell line representing grade II carcinoma) and J82 (Human bladder cancer cell line representing grade III) cells exposed to different MOIs of wild-type BCG. Cell viability was measured using CellTiter-Glo Luminescent Cell Viability assay. Fig. 45A shows cell viability of RT4 cells. Fig. 45B
shows cell viability of 5637 cells. Fig. 45C shows cell viability of J82 cells.
[0067] FIGs 46A-46D show BCG Tice overexpressing c-di-AMP as a stronger inducer of antitumor cytokine response in urothelial carcinoma cells. (A-D) Quantitative measurement of differential TNF-a, IL-6, IL-113 and IFN-y levels using ELISA in different urothelial carcinoma cells 24 h after infection with different wild-type BCG (Tice) and BCG-disA-OE
(Tice) strains.
Cells to BCG infection ratio = 1:20, data represent mean SEM (n = 3 replicates). Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001. Fig. 46A shows TNF-a levels. Fig.
46B shows IL-6 levels. Fig. 46C shows IL-1f3 levels. Fig. 46D shows IFN-y levels.
[0068] FIGs 47A-47G show BCG Pasteur overexpressing c-di-AMP as a stronger inducer of antitumor cytokine response in urothelial carcinoma cells. (A-G) Quantitative measurement of differential cytokine levels using ELISA in different urothelial carcinoma cells 24 h after infection with different wild-type BCG (Pasteur) and BCG-diszl-OE (Pasteur) strains.
Cells to BCG
infection ratio = 1:20. Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001. Fig. 47A shows TNF-a levels in 5637 cells.
Fig. 47B shows TNF-a levels in BBN975 cells. Fig. 47C shows 11-6 levels in UPPL1595 cells.
Fig 47D shows IL-113 levels in MB49 cells. Fig. 47E shows IL-113 levels in UPPL1595 cells. Fig.
47F shows IFN-y levels in BBN975 cells. Fig. 47G shows IFN-y levels in NBT-II
cells.
[0069] FIGs 48A-48B show stronger macrophage reprogramming towards M1 phenotype after infection with BCG strains overexpressing c-di-AMP. Fig. 48A shows wild-type BMDMs infected with different BCG strains and for 24 h at 1:10 MOIs. Cell surface and intracellular straining was carried out and cells were analyzed using flow-cytometry (BD LSR
II flow cytometer. Bar diagram showing percentage of TNE-oc positive antigen presenting mouse macrophages (MHC Class II+CD 1 1b+F4/80+) following infection with wild-type and c-di-AMP
overexpressing BCG Tice and Pasteur strains. Data were processed using FlowJo software (Tree Star v10). Fig. 48B shows representative flow plots showing different cell phenotypes of antigen producing M1 macrophages. Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****13< 0.0001 [0070] FIGs 49A-49B show stronger reprogramming of M2 macrophages after infection with BCG strains overexpressing c-di-AMP. Fig. 49A shows percentage of M2 macrophage surface markers (CD2061CD124' ) positivity on mouse BMDM macrophages (CD1lb F4/80') after infection with wild-type. Fig. 49B shows percentage of IL-10 producing macrophages of M2 macrophage (CD206 CD124+ mouse macrophages) population. Briefly, wild-type BMDMs were generated in presence of murine M-CSF. Macrophages were infected with different BCG strains and for 24h at 1:10 MOIs. Cell surface and intracellular straining were carried out and cells were analyzed using flow-cytometry (BD LSR II flow cytometer. Data were processed using FlowJo software (Tree Star v10). Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0071] FIGs 50A-50B show stronger induction of monocytic MDSCs secreting IL-10 in murine BMDMs after infection with wild-type and c-di-AMP overexpressing BCG
strains. Fig.
50A shows the percentage of M-MDSCs of total myeloid cells (CD45+). Fig. 50B
shows the percentage of IL-10 producing M-MDSCs after infection of murine BMDMs with WT
and BCG-disA-OE strains. Briefly, wild-type BMDM macrophages were infected with different BCG strains and for 24 h at 1:10 MOIs. Cell surface and intracellular straining was carried out and cells were analyzed using flow-cytometry (BD LSR TT flow cytometer. Data was processed using FlowJo software (Tree Star v10). Data represent mean SEM (n = 3 replicates).
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0072] FIGs 51A-51B show differential induction of classical (inflammatory) monocytes after infection with wild-type and c-di-AMP overexpressing BCG Tice. Fig_ 51A shows a bar diagram showing percentage of classical monocytes (CD14 CD16 ) of CD 1 lb populations.
Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors.
Negatively selected human monocytes were infected with wild-type and BCG-disA-OE strains for 24 h (1:10 MOTs). Fig. 51B shows representative flow-cytometry plots showing different percentage of monocyte populations. Cell surface staining was carried out and cells were analyzed using flow-cytomctry (BD LSR 11 flow cytometcr). Data were processed using FlowJo software (Tree Star v10). Student's t-test (two-tailed) *P<0.05, **P<0.01, ***p< P< 0.0001 [0073] FIGs 52A-52B show BCG overexpressing c-di-AMP as a potent inducer of proinflammatory cytokines in human monocyte-derived macrophages. Fig. 52A is a bar diagram showing percentage of MHC class II positive classical macrophages producing TNF-a (TNF-a+HLA-DR+/CD14+CD16 ) and IL-6 (IL-6+HLA-DR+/CD14+CD16 ). Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors. Negatively selected human monocytes were differentiated into macrophages. Macrophages were infected with wild-type and BCG-disA-OE strains for 24 h (1:10 MOIs). Fig. 52B shows representative flow-cytometry plots showing different percentage of macrophage populations. Cell surface staining was carried out and cells were analyzed using flow-cytometry (BD LSR IT flow cytometer. Data were processed using FlowJo software (Tree Star v10). Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0074] FIGs 53A-53C show BCG overexpressing c-di-AMP strongly suppressing M2 macrophage phenotypes. Fig. 53A shows percentage of immunosuppressive M2 (CD206+CD163+) macrophages of total transitional (CD14+CD16+) macrophages.
Fig. 53B
shows percentage of IL-10 producing macrophages of total M2 macrophages (CD206+CD163+) after infection of HMDMs with WT and BCG-disA-OE Tice strains. Fig. 53C shows representative flow-cytometry plots showing M2 cell surface phenotypes and IL-10 producing cells of M2 macrophages. Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors. Negatively selected human monocytes were differentiated into M2 macrophages in presence of M-CSF. Infections were carried out for 24 h (1:10 MOIs), cell surface staining and intracellular staining was performed. Data are mean SEM (n = 3 replicate experiments performed on inonocytes-derived inacrophages from healthy human donors). Student's t-test (two-tailed).
*P<0.05, **P<0.01, ***13<0.001, ****P< 0.0001 [0075] FIGs 54 shows BCG overexpressing c-di-AMP infected macrophages enhanced phagocytosis. HMDMs were infected with WT BCG and BCG-disA-OE strains for 6 h and phagocytic activity was measured by quantifying intracellular FITC-labeled IgG-opsonized latex beads. Images were acquired on live cells. Nuclear staining was done using Hoechst . Image acquisition was carried out using LSM700 confocal microscope at 63X
magnification. Images were process using Fiji software. Cells to BCG infection ratio = 1:10. Data are mean SEM (n =
3 replicate experiments performed on monocytes-derived macrophages from healthy human donors). Student's t-test (two-tailed). *P<0.05, **P<0.01, ***P<0.001, ****P<
0.0001. Fig. 54 is a bar graph showing the quantification of the fluorescence.
[0076] FIGs 55A-55B show BCG overexpressing c-di-AMP as a potent inducer of proinflammatory cytokines in primary human monocytes. (A-B) BCG-dis,4-0E
induces significantly higher gene expression of TNF-a and IL-6 in primary human monocytes as compared to WT BCG. TNF-a and IL-6 expression was accessed in primary human monocytes isolated from different healthy donors using qRT-PCR. RNU6A was used as reference gene, and relative expression was calculated by 2AACT method. Data represent mean SEM (n = 6 different healthy donors). Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P
0.0001. Fig. 55A
shows TNF-a levels. Fig. 55B shows IL-6 levels.
[0077] FIGs 56A-56E show BCG overexpressing c-di-AMP as a stronger inducer of trained immunity epigenetic marks in human monocytes after training. (A-B) Bar diagram showing epigenetic active chromatin mark H3K4me3 on TNF-a and IL-6 gene promoters.
Fold enrichment of epigenetically modified TNF-c. and IL-6 gene promoters in BCG-trained human monocytes isolated from different donors (n = 4 different healthy donors) following re-stimulation by Pam3CSK4. H3K4-trimethylated promoters were enriched and quantified using ChIP-PCR. Fig.
56A shows H3K4me3 levels on TNF-a gene promoter. Fig. 56B shows H3K4me3 levels on TL-6 gene promoter. (C-D) Bar diagram showing epigenetic inactive chromatin mark, H3K9me3 on TNF-a and IL-6 gene promoters. Fold enrichment of epigenetically modified TNF-a and IL-6 gene promoters in BCG-trained human monocytes isolated from different donors (n = 4 different healthy donors) following re-stimulation by Pam3CSK4. H3K9-trimethylated promoters were enriched and quantified using ChIP-PCR. Student's 1-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P 0.0001. Fig. 56C shows H3K9me3 levels on TNF-a gene promoter. Fig.

56D shows H3K9me3 levels on IL-6 gene promoter. Fig. 56E shows schematic representation of ex-vivo BCG training.
[0078] FIGs 57A-57C shows BCG overexpressing c-di-AMP
demonstrating improved antitumor activity in the MN U carcinogen model of NM1BC. Fig. 57A Schematic of intravcsical treatment strategy of BCG in MNU carcinogen model of NMIBC. Fig. 57B is a graph bar showing tumor involvement index. Fig. 57C is a graph showing staging of tumors.
Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****p< 0.0001 [0079] FIGs 58A-58C show BCG-disA-OE strains attenuated for virulence in vivo. Fig. 58A
illustrates BALB/c BCG aerosol challenge model. Fig. 58B shows BALB/c mice lung bacillary burden of wild-type and BCG-disA-OE strains in mouse lungs 4-week post infection. Data are mean S.E.M. (n = 5 animals/group). Fig. 58C shows implantation (day 01) of BCG strains following aerosol challenge in BALB/c mice. Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0080] FIGs 59A-59C show BCG strain overexpressing c-di-AMP
attenuated for virulence in a severely immunocompromised (SCID) mouse model of aerosol infection. Fig. 59A
illustrates SCID mice model of BCG aerosol infection. Fig. 59B shows survival of SCID mice (n =10) after infection with different BCG strains. Fig. 59C shows implantation (day 01) of BCG strains following aerosol challenge in SCID mice. Student's 1-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0081] FIGs 60A-60D show BCG overexpressing c-di-AMP as a stronger inducer of Thl cytokines in vivo. (A-B) Quantitative levels of IFN-P, TNF-a, IL-6 and IFN-y in lung homogenates from BALB/c mice at 4 weeks after infection with different BCG strains using ELISA. Fig. 60A
shows results with WT BCG (Tice) and BCD-disA-OE (Tice). Fig. 60B shows results with WT
BCG (Pasteur) and BCD-disA-OE (Pasteur). (C-D) Quantitative levels of IFN-p, TNF-a, IL-6 and IFN-y in lung homogenates from BALB/c mice at 4 weeks after infection with different BCG
strains using ELISA. Fig. 60C shows results with WT BCG (Tice) and BCD-disA -OE (Tice). Fig.
60D shows results with WT BCG (Pasteur) and BCD-disA-OE (Pasteur). Results are represented as the mean (pg/ml) SEM (n = 4 animals/group). Student's t-test. Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P< 0.0001 [0082] FIGs 61A-61B show therapeutic intratumoral injection of BCG overexpressing c-di-AMP leading to greater antitumor activities in MB49 model of bladder cancer.
Fig. 61A shows a schematic representation of intratumoral injection of tumors. Mice were implanted with lx105 MB49 cells on day 0, then accessed for tumor volume until the group averaged ¨40mm3. At that time, mice were treated with 5x106 wild-type or BCG-disA-OE strain in a total volume of 50 L
or with PBS alone every 3rd day for a total of 3 treatments. Fig. 61B shows tumor outgrowth of MB49 bearing animals treated with vehicle (PBS), wild-type and BCG-disA -OE
following treatments as shown in figure 61A. Two-way ANOVA.
[0083] FIGs 62A-62D show BCG overexpressing c-di-AMP inducing stronger infiltration of IFN-y at tumor site following intratumoral administration. (A-B) Dot plot showing relative abundance of percentage of CD4 or CD8 of total CD3 populations in single cells isolated from tumor. Fig. 62A shows percentage of CD4 cells. Fig. 62B shows percentage of CD8 cells. (C-D) Dot plot showing higher percentage of interferon-y producing CD4 T cells inside single cells isolated from tumors receiving BCG-disA -OE. Fig. 62C shows percentage of INF-y+ cells among CD4 cells. Fig. 6211 shows percentage of INF-y cells among CD8 cells. No significant changes in percentage of interferon-y producing CD8 T cells were observed. Briefly, single cells were prepared from excised tumors after intratumoral administration of PBS or BCG
strains. Cell surface and intracellular cytokine staining was performed, and cells were analyzed using flow cytometry. Student's t-test. Student's t-test (two-tailed) *P<0.05, **P<0.01, ***P<0.001, ****P<
0.0001 DETAILED DESCRIPTION OF THE INVENTION
[0084] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994);
The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &
Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
[0085] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
[0086] By "alteration" is meant a change (increase or decrease) in the expression level or activity of a gene or polypeptide as detected by standard methods known in the art such as those described herein. As used herein, an alteration includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression level.
[0087] By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
[0088] By "analog" is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could, for example, increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid, in another example.
[0089] By "cdnP" is meant either 1) a cdnP gene or nucleic acid sequence that encodes a cyclic di-nucleotide phosphodiesterase (cdnP) protein or 2) the cyclic di-nucleotide phosphodiesterase protein. Examples include the AI tuberculosis cdnP gene in H37Rv, Rv2837c, having NCBI Gene ID 888920, and a cdnP protein of UniProtKB/Swiss-Prot P71615.2.
100901 By "cGas" is meant either 1) a cGas gene or nucleic acid sequence that encodes a cyclic GMP-AMP synthase (cGAS) protein, or 2) the cyclic GMP-AMP synthase protein.
Examples of cGas include the H. sapiens cGAS gene (NCBI Gene ID: 115004) and the protein encoded by this gene (UniProtKB/Swiss-Prot: Q8N884.2). The cGas protein is a cyclic GMP-AMP
synthase from humans that makes 2'3' cGMP. 2'3' cGMP is a STING agonist in humans.
[0091] "Cyclase domains" of cGAS for example, refers to a portion or fragment of the 522 amino acids of the human cGAS protein described in Kranzusch et al. (Cell Reports 2013; 3:1362-1368 PMID 23707061). A cyclase domain may be described as having an NTase core situated from amino acid 160-330, and a regulatory-sensor domain that is the C-domain situated from amino acids 330-522. Mutants of the NTase core sequence as well as mutants of the regulatory-sensor domain can be used to generate constitutively active variants of cGAMP
designed to produce high levels of cGAMP without the normal requirement for activation by DNA binding.
Another example of a cyclase domain includes M. tuberculosis Rv1354c of NCBI
Gene ID:
887485, and the protein encoded by this gene (UniProtl(B/Swiss-Prot: P9WM13) that encodes a 623 amino acid-long protein capable of both c-di-GMP (cyclic diguanylate or cyclic di-GMP) synthesis (via its GGDEF domain, amino acids 201-400) and degradation (via its EAL domain, amino acids 401-623). The GAF domain (amino acids 1-200) is a regulatory domain. The GGDEF
domain as well as mutants of the regulatory-sensor GAF domain and polypeptides truncated to remove the EAL domain (phosphodiesterase activity) can be used to generate constitutively active variants of Rv1354c designed to produce high levels of c-di-GMP.
[0092] By "DisA" or "disA" is meant either 1) a Dis A gene or nucleic acid sequence that encodes a DNA integrity scanning (DisA) protein or 2) the DNA integrity scanning protein.
Examples include M. tuberculosis disA gene Rv3586 of NCBI Gene ID: 887485, and the protein encoded by this gene is UniProtKB/Swiss-Prot: P9WNW5.1. The protein is a 358 amino acid-long diadenylate cyclase as described by Dey & Bishai et al. Nature Medicine 2015;21:401-6. PMID:
25730264. A DisA protein is a diadenylate cyclase that makes c-di-AMP. c-di-AMP is a STING
agoni st.
[0093] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include, but are not limited to, bladder cancer.
100941 By "dncV" is meant a gene that encodes a Cyclic GMP-AMP
synthase that catalyzes the synthesis of 3'3'-cyclic GMP-AMP (3'3'-cGAMP) from GTP and ATP, a second messenger in cell signal transduction. dncV isalso able to produce c-di-AMP and c-di-GMP
from ATP and GTP, respectively; however, 33-cGAMP is the dominant molecule produced by DncV in vivo, contrary to the 2'3'-cGAMP produced by eukaryotes. dncV isrequired for efficient V.cholerae intestinal colonization, and down-regulates the colonization-influencing process of chemotaxis. dncV is not active with dATP, TTP, UTP, and CTP. The DncV protein is a cyclic GMP-AMP
synthase from V.cholerae that makes 3'3'cGAMP. 3'3'cGAMP is a STING agonist.

[0095] -EAL domain" means a conserved protein domain that is found in diverse bacterial signaling proteins. The EAL domain may function as a diguanylate phosphodiesterase and has been shown to stimulate degradation of a second messenger, cyclic di-GMP. A
non-functional EAL domain will not have one or more of these functions. An example of an EAL
domain includes M tuberculosis Rv1357c gene of NCBI Gene ID: 886815, and the 307 amino acid-long protein encoded by this gene is UniProtKB/Swiss-Prot: P9WM07 that encodes a c-di-GMP
phosphodiesterase (PDE) and is comprised of a sole EAL domain. This enzyme's activity is to serve as a c-di-GMP phosphodiesterase, cleaving the cyclic dinucleotide (which has signaling activity) into 2 GMP molecules (which lack signaling activity), as described in the article titled, "A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis," Bharati BK, Sharma IM, Kasetty S, Kumar M, Mukherjee R, Chatterji D. Microbiology. 2012 Jun;158(Pt 6):1415-27. doi:

10.1099/micØ053892-0. Epub 2012 Feb 16.PMID: 22343354. Another example of an EAL
domain includes the 336 amino acid-long protein encoded by Al. tuberculosis cdnP gene in H37Rv (Rv2837c), a c-di-AMP phosphodiesterase comprising an EAL domain with the capability of hydrolyzing human 2'-3'cGAMP (the product of the human cGAS enzyme) as shown by Jain-Dey Bishai et al. Nat Chem Biol. 2017;13:210-217 PMID 28106876. The structural characteristics of the EAL domains (cyclic dinucleotide phosphodiesterase activity) and GGDEF
domains (cyclic dinucleotide cyclization-biosynthetic activity) are known and well described (for example, in Schirmer T, Jenal U. Structural and mechanistic determinants of c-di-GMP
signaling. Nat Rev Microbiol. 2009;7:724-35. PMID: 19756011).
[0096] By "effective amount" is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
[0097] By -dncV" is meant either 1) a dncV gene or nucleic acid sequence that encodes a cyclic GMP-AMP synthase (DncV) protein, or 2) the Cyclic GMP-AMP synthase protein.

Examples include, but are not limited to, the Vibrio cholerae dncV gene of NCBI Gene ID:
2614190 and the protein encoded by this gene is UniProtKB/Swiss-Prot: Q9KVG7.1 [0098] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 300/u, 40%, 50%, 60%, 70%, 800/u, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A
fragment may contain, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
[0099] By "gene deletion" is meant using allelic exchange methodologies well-known to one skilled in the art to delete the full gene coding region of the gene of interest from the chromosome of BCG. Gene replacement with selectable markers such as antibiotic resistance cassettes is a form of allelic exchange and may be performed. Technologies are also available to generate unmarked deletions (no selectable marker) in which the gene is entirely deleted, and no selectable marker is introduced in its place.
[0100] By "gene domain deletion" is meant using the above allelic exchange methodologies to remove the portion of a gene encoding a particular domain (in the case of the present invention the EAL domain of Rv1354c which encodes the CDN phosphodiesterase domain of a multifunctional polypeptide) leaving the other portions of the polypeptide intact and in frame.
[0101] By "H. sapiens" is meant Homo sapiens.
[0102] By "obtaining" as in "obtaining an agent" is meant synthesizing, purchasing, or otherwise acquiring the agent.
[0103] By "overexpression" is meant, in a general sense, a gene expressing its corresponding protein in a greater quantity than a wild type or reference gene. An example of creating a gene overexpressing a protein in the present invention includes fusing the DNA
encoding the gene of interest to a strong promoter in BCG such as Phsp60 or to a strong conditionally active promoter such as PtetOFF. In PtetOFF, gene expression is turned off in the presence of tetracycline, anhydrotetracycline, or doxycycline; however, when the recombinant BCG is administered as an immunotherapy in a human or an animal model, the gene of interest will be turned on. This conditionally active strategy has the advantage of preventing any deleterious effects on viability or growth rate that strong overexpression of cyclic dinucleotide producing enzyme might have on the BCG organisms while the BCG is being grown, and it allows for strong expression ("overexpression") only when the BCG immunotherapy is given as a therapeutic to a mammalian host.
[0104] By "Mib" is meant Mycobacleriurn lubercitlosis.
[0105] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide"
and "protein" include glycoproteins, as well as non-glycoproteins.
[0106] By "reduce" or "decrease" is meant a negative alteration of at least about 100/u, 25%, 50%, 75%, or 100%, for example, or any percentage in between.
[0107] By "increase" is meant a positive alteration of at least about 10%, 25%, 50%, 75%, or 100%, for example, or any percentage in between.
[0108] By "reference" is meant a standard or control condition.
[0109] A "reference sequence" is meant a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence, for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or there between.
[0110] By "reference BCG strain" is meant, for example, a conventional BCG strain that does not contain the expression vectors of the present invention and/or the endogenous genes unable to express a cdnP functional protein, a Rv1354c functional protein, a Rv1357c functional protein, or a combination thereof.
[0111] By "regulatory DNA recognition capability" is meant the ability of a protein to detect or bind DNA. For example, a cGAS protein is known to bind DNA, such as cytosolic DNA, and triggers the reaction of GTP and ATP to form cyclic GMP-AMP (cGAMP). cGAMP
binds to the Stimulator Interferon Genes (STING) which triggers phosphorylation of IRF3 via TBK1.
[0112] By "Rv1354c" is meant either 1) a Rv1354c gene or nucleic acid sequence that encodes a Rv1354c protein or 2) the Rv1354c protein (e.g., Gupta, Kumar, and Chatterji; PLoS ONE
(November, 2010); Vol. 5; Issue 11; and Bhariati, Sharma, Kasetty, Kumar, Mukherjee, and Chatterji; Microbiology (2012), 158, 1415-1427). The Rv1354c protein is a diguanylate cyclase that mkes c-di-GMP. C-di-GMP is a STING agonist.
[0113] By "Rv1357c" is meant either 1) a Rv1357 gene or nucleic acid sequence that encodes a cyclic di-GMP phosphodiesterase protein (Rv1357) protein or 2) the cyclic di-GMP
phosphodiesterase protein (e.g., Gupta, Kumar, and Chatterji; PLoS ONE
(November, 2010); Vol.
5; Issue 11; and Bhariati, Sharma, Kasetty, Kumar, Mukherjee, and Chatterji;
Microbiology(2012), 158, 1415-1427). The Rv1357c protein is a diguanylate cyclase that mkes c-di-GMP. C-di-GMP
is a STING agonist.
[0114] By "STING agonist" is meant a molecule which binds to STING (stimulator of interferon genes, or TMEM173), activates it, and triggers activation of the IRF3-TBK1 pathway leading to increased transcription of type 1 interferon and other genes.
[0115] By -CDN" is meant cyclic dinuculeotide such as 3'-5' c-di-AMP, 3'-5' c-di-GMP, 3'-3' cGAMP (also known as 3'-5', 3'-5'cGAMP, the product of the Vibrio eholerae DncV
protein), or 2'-3' cGAMP (also known as 2'-5', 3'-5' cGAMP, the product of the human cGAS
protein).
[0116] By -PAMP" is meant pathogen associated molecular pattern.
PAMPs are microbial products including small molecules which are recognized by innate immune sensors. Examples of PAMPs are 3'-5' c-di-AMP, 3'-5' c-di-GMP, 3'-3' cGAMP.
[0117] By "DAMP" is meant danger associated molecular pattern.
DAMPs are host-derived (that is human, mouse, or other mammalian model of disease) molecules that are produced to signal danger such as infection or other derangement of normal physiology. An example of a DAMP is 2'-3' cGAMP which is produced by the host sensor enzyme cGAS upon detection of double-stranded DNA in the cytosol as occurs during viral or certain intracellular bacterial infections.
[0118] By "panCD" is meant the genetic operon from bacteria or other species the encodes the biosynthetic gene panC (encoding the PanC protein which has pantoate-beta-alanine ligase enzymatic activity) and the biosynthetic gene panD (encoding the PanD protein which has aspartate 1-decarboxylase enzymatic activity). The PanC and PanD proteins are required for the biosynthesis of pantothenic acid or pantothenate also called vitamin B5 (a B
vitamin). Pantothenic acid, a water-soluble vitamin, is an essential nutrient for bacteria and for all mycobacteria including BCG. Pantothenic acid is required in order to synthesize coenzyme-A (CoA), as well as to synthesize and metabolize proteins, carbohydrates, and fats.
[0119] By "specifically binds" is meant a compound, nucleic acid, peptide, protein, or antibody, for example, that recognizes and binds a polypeptide or nucleic acid sequence, but which does not substantially recognize and bind other molecules in a sample.
[0120] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.
53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; senile, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-m indicating a closely related sequence.
[0121] By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
[0122] By "sensitivity" is meant the percentage of subjects with a particular disease.
[0123] By -specificity" is meant the percentage of subjects correctly identified as having a particular disease, i.e., normal or healthy subjects. For example, the specificity is calculated as the number of subjects with a particular disease as compared to normal healthy subjects (e.g. ,non-cancer subjects).
[0124] By "trained immunity" is meant the ability of one antigenic stimulus to elicit more potent immune responses to a second, different antigen administered at a later time. Trained immunity is antigen-independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and is associated with epigenetic and metabolic changes.
[0125] By "Phsp60" or "Phsp65" is meant a strong mycobacterial promoter derived from the Mycobacterium leprae Hsp65 5'UTR.
[0126] By "5'UTR" is meant the 5' untranslated region of a gene.
[0127] By "3'UTR" is meant the 3' untranslated region of a gene.
[0128] By "WT" is meant wild type.
[0129] By "BCG-WT" is meant a wild type strain of Mycobacterium bovis bacillus Calmette Guerin.
[0130] Ranges provided herein are to be understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is to be understood to include any number, combination of numbers, or sub-range from the group consisting of 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
[0131] As used herein, the terms "treat," "treating,"
"treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
101321 Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.
[0133] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0134] As used herein, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes,"
"including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
[0135] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
[0136] As used herein, the terms "prevent," "preventing,"
"prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
[0137] Such treatment (surgery and/or chemotherapy) will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for bladder cancer or disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, a marker (as defined herein), family history, and the like). In some embodiments, determination of subjects susceptible to or having a urothelial cancer is determined by measuring levels of at least one of the markers.
[0138] 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 p-hagocyti c cells such as macrophages, den dri ti c 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 phosphodi esterase genes or genetic domains that will enhance the accumulation and release of CDNs.
[0139] BCG

[0140] 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.
[0141] 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.
[0142] 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 (IFNy) in the bladder, and (iii) increase urinary cytokine levels of IL-1, IL-2, IL-6, IL-8, IL-12, 1FNy, and TNFa.
[0143] 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.
[0144] Urothelial cancer [0145] Urothclial 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 Ti), known 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.
[0146] 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 EJ, Newton MR, O'Donnell MA, 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 immunothcrapy. 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, remains unclear, but it is believed to orchestrate a vigorous immune cellular and humoral immune response, predominantly Th 1 response, after binding to the urothelium through fibronectin and integrin a5131 (Redelman-Sidi G, Glickman MS, Bochner BH.
The mechanism of action of BCG therapy for bladder cancer--a 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). (A
skel and ET, Newton MR, O'Donnell MA, Luo Y. Bladder Cancer Immunotherapy: BCG and Beyond. Adv Urol.
2012;2012:181987. PMED: 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.
PM1D: 28646932). The burden of patients with BCG unresponsive and relapsing disease and of those intolerant to treatment has therefore prompted the need for further improving the efficacy of BCG against NMIBC.
[0147] CDNs are important PAMPs and DAMPs that generate valuable immune responses for TB and NMIBC.
[0148] 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 NFicB through the IicB kinase (IKK) activation.
[0149] 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 2'3' cGAMP. 2'3' 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.
[0150] 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 0, Smyth MJ, 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 eytosolic 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 to and activates STING. (Zhao GN, Jiang DS, 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.
101511 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, 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 Thl, Th17 and CD8 T cell responses against pathogens. A c-di-AMP¨overexpressing BCG strain (rBCG-disA or BCG-disA-0E) has been constructed and it was surprisingly found that it produced a significantly higher 1RF and IFN-13 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 [0152] Induction of pro-inflammatory Thl cytokines in mouse bone marrow-derived macrophages (BMDMs) in response to BCG oyerexpressing nth disA (MT3692): Mil) genome encodes a di-adenylate cyclase enzyme (DisA, also called DacA, P9WNW5.1 in the UniProtKB/Swiss-Prot databases) that synthesizes e-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-13 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, Phsp60. Addition of this plasmid to BCG-Pasteur increased the level of disA mRNA by 50-fold (Fig. lb). 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. la), 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-0E) were better inducers of STING-dependent IFN-(3 as compared to the parental strain. Most importantly as reported in PCT/US2016/017248, filed February 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.
[0153] 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-f3, TNF-a, IL-6 and IL-2 in comparison to uninfected or wild-type BCG infected macrophages.
[0154] 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-0E, as compared to parental control.
[0155] As shown in Figs 4A-4C, a significant increase in secreted pro-inflammatory cytokines (TNF-a, 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 T IFNs.
Furthermore, the immune responses in macrophages in response to BCG-Pasteur disA-OE were skewed towards Thl, a phenotype largely attributed for control of NMIBC by BCG

immunotherapy.
[0156] 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 3h, and differential gene expression profile was determined in comparison to uninfected cells. Key immune mediators such as, monocyte chemoattractant protein 1 (MCP-1)/CCL2, TEN-13 and IL-113 were found to be significantly increased in bladder cancer cells exposed to BCG-Pasteur-disA-OE
compared to responses to wild type strains.
[0157] As shown in Fig. 6, an experimental system was set up to test whether intravesical BCG-disA-OE immunotherapy leads to heightened Thl 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 di sA OE in an in vivo rat NMIBC model, pioneered in Bivalacqua lab. (Kates M, Nirschl T, Sopko NA, Matsui H, Kochel CM, Reis LO, Netto GJ, Hogue MO, Hahn NM, McConkey DJ, Baras AS, Drake CG, Bivalacqua TJ. 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.
[0158] 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 Ti urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human urothelial cancer. Using this model, it was showed that intravesical BCG immunotherapy lead to a large, transient rise in the CD4 T cell population in the urothelium. (Kates M, Nirschl T, Sopko NA, Matsui H, Kochel CM, Reis LO, Netto GJ, Roque MO, Hahn NM, McConkey DJ, Baras AS, Drake CG, Bivalacqua TJ.
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, Ti and CIS) calculated for each group according to criteria as described.
(Kates M, Nirschl T, Sopko NA, Matsui H, Kochel CM, Reis LO, Netto GJ, Hogue MO, Hahn NM, McConkey DJ, Baras AS, Drake CG, Bivalacqua TJ. 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.
[0159] 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-a/[3, IFN-y, IL-113, INF-a, IGF-Pp, iNOS, IP-10, MCP-1 and MIP-la in comparison to untreated or BCG-Pasteur treated rats.
[0160] As shown in Fig. 9, evidence was found for increased infiltration of CCL2+
macrophages, Nos2 and IL-113' M1 macrophages, accompanied by increased IL-6 and IFN-expression in bladders of rats treated with BCG-Pasteur-disA-0E.
Interestingly, increased levels of IP-10 were found, which together with increased IFN-y is known to promote a strong T cell recruitment at the site of infection and inflammation.
[0161] 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 Thl T cell and M1 macrophage expansion, two type 1 interferons, and three pro-inflammatory chemokines were significantly upregulated 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).

[0162] BCG immunotherapy may be effective via three immune mechanisms: (i) increased generation of tumor-specific cytotoxic CDS T cells, (ii) cytokine environment which promotes macrophage-mediated CD4 cell activation against tumor antigens, and (iii) macrophage MI 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.
[0163] 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. Genes for other CDN
cyclases--(i) the GGDEF domain of the BCG_R507340 protein or /14. tuberculosis Rv1354c protein (100% identical to each other), (ii) the Vibrio cholerae DncV protein, Q9KVG7 in Swiss-Prot, which is a 2'-5'c-GAMP synthase, and (iii) the human cGAS protein Q8N884 in Swiss-Prot which is a 2'-3' cGAMP synthase--may 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. 11, 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.
[0164] SEQ ID NO:1 [0165] Diadenylate cyclase DisA from BCG and other related mycobacteria, amino acid sequence (358 amino acids; BCG protein A0Q92_RS18745; NCBT 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.
MHAVTRPTLREAVARLAPGTGLRDGLERILRGRTGALIVLGHDENVEATCDGGESLDVRY
AATRLRELCKMDGAVVLSTDGSRIVRANVQLVPDPSIPTDESGTRHRSAERAATQTGYPV

VTLRDVMTVVQRLELVRRIGLVIDYDVVELGTDGRQLRLQLDELLGGNDTARELIVRDYH
ANPEPPSTGQINATLDELDALSDGDLLDFTALAKVFGYPTTTEAQDSTLSPRGYRAMAGI
PRLQFAHADLLVRAFGTLQGLLAASAGDLQSVDGIGAMWARHVREGLSQLAESTISDQ
[0166] SEQ ID NO:2 [0167] Diadenylate cyclase disii from BCG and other related mycobacteria, DNA sequence (1077 nucleotides 1358 codons, 1 stop codonl; 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.
1 atgcacgctg tgactcgtcc gaccctgegt gaggctgtcg cccgcclagc cccgggcact 61 gggctgcggg acggcctgga gcgtatcctg cgcggccgca ctggtgccct gatcgtgctg 121 ggccatgacg agaatgtcga ggccatctgc gatggtggct tctccctcga tgtccgctat 181 gcagcaaccc ggctacgcga gctgtgcaag atggacggcg ccgtggtgct gtccaccgac 241 ggcagccgca tcgtgcgggc caacgtgcaa ctggtaccgg atccgtcgat ccccaccgac 301 gaatcgggga cccggcaccg ctcggccgag cgggccgcga tccagaccgg ttacccggtg 361 atctcagtga gccactcgat gaacatcgtg accgtctacg tccgcgggga acgt, cacgta 421 ttgaccgact cggca.accat cctgtcgcgg gccaaccagg ccatcgcaac cctggagcgg 481 tacaaaacca ggctcgacga ggtcagccgg caactgtcca gggcagaaat cgaggacttc 541 gtcacgctgc gcgatgtgat gacggtggtg caacgcctcg a.gctggfccg gcga.atcggg 601 ctggtgatcg actacgacgt ggtcgaactc ggcactgatg gtcgtcagct gcggctgcag 661 ctcgacgagt tgctcggcgg caacgacacc gcccgggaat tgatcgtgcg cgattaccac 721 gccaacccgg aaccaccgtc cacggggcaa atcaatgcca ccctggacga actggacgcc 781 ctgtcggacg gcgacctcct cgatttcacc gcgctggcaa aggttttcgg atatccgacg 841 accacggaag cgcaggattc ggcgctgagc ccgcgtggct accgcgcgat ggccggtatc 901 ccccggctcc agttcgccca tgccgacctg ctggtccggg cgttcggaac gttgcagggt 961 ctgctggcgg ccagcgccgg cgatctgcaa tcagtggacg gcatcggcgc catgtgggcc 1021 cgtcatgtgc gcgatgggtt gtcacagctg gcggaatcga ccatcagcga tcaataa [0168] SEQ ID NO:3.
[0169] Plasmid pSD5B-Phsp6o::dis,4 is an episomally replicating E. coli-mycobacterial shuttle plasmid that overexpresses the BCG disil gene from the Phsp60 promoter, DNA
sequence. (7742 nucleotides; promoter Phsp6o DNA comprised of a portion of the M. leprae hsp65 gene nucleotides 13 to 180 is underlined; disA coding sequence nucleotides 242 to 131 g; ATG
start codon and TAA
stop codon shown in boldface, underline).
GGATCCTTCTAGAATTCCGGAATTGCACTCGCCTTAGGGGAGTGCTAAAAATGATCCTGGCACTCGCGATCAGCGAG

TGCCAGGTCGGGACGGTGAGACCCAGCCAGCAAGCTGTGGTCGTCCGTCGCGGGCACTGCACCCGGCCAGCGTAAGT

AATGGGGGTTGTCGGCACCCGGTGACCTAGACACATGCATGCATGCTTAATTAATTAAGCGATATCCGGAGGAATCA

CTTCCATATGATCCACGCTGTGACTCGTCCGACCCTGCGTGAGGCTGTCGCCCGCCTAGCCCCGGGCACTGGGCTGC

GGGACGGCCTGGAGCGTATCCTGCGCGGCCGCACTGGTGCCCTGATCGTGCTGGGCCATGACGAGAATGTCGAGGCC

ATCTGCGATGGTGGCTTCTCCCTCGATGTCCGCTATGCAGCAACCCGGCTACGCGAGCTGTGCAAGATGGACGGCGC

CGTGGTGCTGTCCACCGACGGCAGCCGCATCGTGCGGGCCAACGTGCAACTGGTACCGGATCCGTCGATCCCCACCG

ACGAATCGGGGACCCGGCACCGCTCGGCCGAGCGGGCCGCGATCCAGACCGGTTACCCGGTGATCTCAGTGAGCCAC

TCGATGAACATCGTGACCGTCTACGTCCGCGGGGAACGTCACGTATTGACCGACTCGGCAACCATCCTGTCGCGGGC

CAACCAGGCCATCGCAACCCTGGAGCGGTACAAAACCAGGCTCGACGAGGTCAGCCGGCAACTGTCCAGGGCAGAAA

TCCiAGGACTTCGTCACGCTGCGCGATCiTGATGACCiGTGGTGCAACGCCTCCiAGCTGGTCCGGCGAATCGGGCTli ATCGACTACGACGTGGTCGAACTCGGCACTGATGGICGTCAGCTGCGGCTGCAGCTCGACGAGTTGCTCGGCGGCAA

CGACACCGCCCGGGAATIGATCGIGCGCGATTACCACGCCAACCCGGAACCACCGICCACGGGGCAAATCAATGCCA

CCCIGGACGAACTGGACGCCCIGICGGACGGCGACCICCTCGATTICACCGCGCIGGCAAAGGTTITCGGATATCCG

ACGACCACGGAAGCGCAGGATTCGACGCTGAGCCCGCGTGGCTACCGCGCGATGGCCGGTATCCCCCGGCTCCAGTT

CGCCCATGCCGACCTGCTGGTCCGGGCGTTCGGAACGTTGCAGGGTCTGCTGGCGGCCAGCGCCGGCGATCTGCAAT

CAGTGGACGGCATCGGCGCCATGTGGGCCCGTCATGTGCGCGAGGGGTTGTCACAGCTGGCGGAATCGACCATCAGC

GATCAATAAACGCGTTCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT

GGCGAATGGCGCTTTGCCTGGTTTCCGGTCGAAGCTTGGCCGGATCTAAAGTTTTGTCGTCTTTCCAGACGTTAGTA

AATGAATTTTCTGTATGAGGTTTTGCTAAACAACTTTCAACAGTTTCAGCGGAGTGAGAATAGAAAGGAACAACTAA

AGGAATTGCGAATAATAATTTTTTCACGTTGAAAATCTCCAAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGTATC

GGTTTATCAGCTTGCTTTCGAGGTGAATTTCTTAAACAGCTTGATACCGATAGTTGCGCCGACAATGACAACAACCA

TCGCCCACGCATAACCGATATATTCGGTCGCTGAGGCTTGCAGGGAGTCAAAGGCCGCTTTTGCGGGGATCCGCTC1i GAGGCGCGGICGCGGCGCGGCTGTGGCATGTCGGGGCGTGCCGCTCCECCGGCGCCGCCCATCGGCCCGCCCATTGG

CATICCGCCCATGCCGCCCATCATICCIGIGGAGCCAGAACTGATCCAGCCIGIGCCACAGCCGACAGGATGGIGAC

CACCATTTGCCCCATATCACCGTCGGTACTGATCCCGTCGTCAATAAACCGAACCGCTACACCCTGAGCATCAAACT

CTTTTATCAGTTGGATCATGTCGGCGGTGTCGCGGCCAAGACGGTCGAGCTTCTTCACCAGAATGACATCACCTTCC

TCCACCTTCATCCTCAGCAAATCCAGCCCTTCCCGATCTGTTGAACTGCCGGATGCCTTGTCGGTAAAGATGCGGTT

AGCTTTTACCCCTGCATCTTTGAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAAT

CGCCCCATCATCCAGC CAGAAAG TGAGGGAGC CAC GG TTGATGAGAGCTTTGTTG TAGG

TGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTT

CGATTTATTCAACA A AGCCGCCGTCCCGTCAAGTCAGCGTA ATGCTCTGCCACiTGTTACA ACCA ATT A

GATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAA

AAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA

TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCA

TGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATT

ACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGC

GATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGC GCAGGAACACTGCCAGCGCATCAACAATA

TTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGC

ATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCT

CATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT

CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGA

ATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAG

CAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGG

CTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGT

GGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCT

GGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTAGCGli AGTGTATACTOGCTTACTATGTTGOCACTGATGAGGGTGTCAGTOA AGTGCTTCATOT(1( i(1,Ati(iA( 1A,A AA AAtiCiCTri 3620-3696 CGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTC CTGGAAGATGCCAGGAAGATACTTAACAGGGA

AGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAA

TCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTG

TTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTC

CGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTA

ACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGA

GTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGT

TACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAG

CAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAA

CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAAGTGCTCATCATTG

GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA

CCCA A CTGA TCTTCA CiCA TCTTTTAC TTTC ACC AGCCITTTCTGCiGTGA GCA A AA ACAGGA
ACICiCA A A A TGCCGCA A A 4621-4697 AAAGGGAATAAGGGCGACACGGAAATGITCiAATACTCATACTCTICCTTITTCAATATTATTGAAGCATTTATCACiG

GrlATTGICTCATGAGCGGATACATAFFYGAATGTATITAGAAAAATAAACAAATAGGGGITCCGCGCACATFICCC

CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCC

CTTTCGTCTTCAAGAATTCCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTG

TTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCG

GAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGGAATTCCCATCGAGCCGAGAACGTTATCGAAGTTGGTCA

TGTGTAATCCCCTCGTTTGAACTTTGGATTAAGCGTAGATACACCCTTGGACAAGCCAGTTGGATTCGGAGACAAGC

AAATTCAGCCTTAAAAAGGGCGAGGCCCTGCGGTGGTGGAACACCGCAGGGCCTCTAACCGCTCGACGCGCTGCACC

AACCAGCCCGCGAACGGCTGGCAGCCAGCGTAAGGCGCGGCTCATCGGGCGGCGTTCGCCACGATGTCCTGCACTTC

GAGCCAAGCCTCGAACACCTGCTGGTGTGCACGACTCACCCGGTTGTTGACACCGCGCGCGGCCGTGCGGGCTCGGT

GOGGCGGCTCTGTCGCCCTTGCCAGCGTGAGTAGCGCGTACCTCACCTCGCCCAACAGGTCGCACACAGCCGATTCG

TACGCCATAAAGCCAGGTGACiCCCACCAGCTCCGTAAGTTCGGGCGCTGTGTGGCTCCiTACCCGCGCATTCAGGCGG

CAGGGGGTCTAACGGGICLAAGGCGGCCiTGTACGCGGCCACAGCGGCTCTCAGCGGCCCGGAAACGTCCTCGAAACG

ACGCATGIGTICCTCCTGGITGGTACAGGIGGTIGGGGGTGCTCGGCTGICGCGGTTGTICCACCACCAGGGCTCGA

CGGGAGAGCGGGGGAGTGTGCACTTGTGGGGTGGCCCCTCAGCGAAATATCTGACTTGGAGCTCGTGTCGGACCATA

CACCGGTGATTAATCGTGGTCTACTACCAAGCGTGAGCCACGTCGCCGACGAATTTGAGCAGCTCTGGCTGCCGTAC

TGGCCGCTGGCAAGCGACGATCTGCTCGAGGGGATCTACCGCCAAAGCCGCGCGTCGGCCCTAGGCCGCCGGTACAT

CGAGGCGAACCCAACAGCGCTGGCAAACCTGCTGGTCGTGGACGTAGACCATCCAGACGCAGCGCTCCGAGCGCTCA

GCGCCCGGGGGTCCCATCCGCTGCCCAACGCGATCGTGGGCAATCGCGCCAACGGCCACGCACACGCAGTGTGGGCA

CTCAACGCCCCTGTTCCACGCACCGAATACGCGCGGCGTAAGCCGCTCGCATACATGGCGGCGTGCGCCGAAGGCCT

TCGGCGGCCGTCGACGGCGACCGCAGTTACTCAGGCCTCATGACCAAAAACCCCGGCCACATCGCCTGGGAAACGGA

ATGGCTCCACTCAGATCTCTACACACTCAGCCACATCGAGGCCGAGCTCGGCGCGAACATGCCACCGCCGCGCTGGC

GTCAGCAGACCACGTACAAAGCGGCTCCGACGCCGCTAGGGCGGAATTGCGCACTGTTCGATTCCGTCAGGTTGTGG

GCCTATCGTCCCGCCCTCATCiCGGATCT ACCTGCCG ACCCGG A A CG TGG A CGG A CTCGGCCGCGCG

GTGCCACGCGCGAAACGCCGAATTCCCGTGCAACGACGTGTGTCCCGGACCGCTACCGGACAGCGAGGTCCGCGCCA

TCGCCAACAGCATITGGCGTIGGATCACAACCAAGTCGCGCATTIGGGCGGACGGGATCGIGGICIACGAGGCCACA

CICAGTGCGCGCCAGICGGCCATCTCGCGGAAGGGCGCAGCAGCGCGCACGGCGGCGAGCACAGITGCGCGGCGCGC

AAAGTCCGCGTCAGCCATGGAGGCATTGCTATGAGCGACGGCTACAGCGACGGCTACAGCGACGGCTACAACCGGCA

GCCGACTGTCCGCAAAAAGCCGTGACGCGCCGAAGGCGCTCGAATCACCGGACTATCCGAACGCCACGTCGTCCGGC

TCGTGGCGCAGGAACGCAGCGAGTGGCTCGCCGAGCAGGCTGCACGCGCGCGAAGCATCCGCGCCTATCACGACGAC

GAG GGCCACTCTTGGCCGCAAACGGCCAAACATTTCGGGCTGCATCTGGACACCGTTAAGCGACTCGGCTATCGGGC

GAGGAAAGAGCGTGCGGCAGAACAGGAAGCGGCTCAAAAGGCCCACAACGAAGCCGACAATCCACCGCTGTTCTAAC

GCAATTGGGGACGGGTGTCGCGGGGGTTCCGTGGGGGGTTCCGTTGCAACGGGTCGGACAGGTAAAAGTCCTGGTAG

ACGCTAGTTTTCTGGTTTGGGCCATGCCTGTCTCGTTGCGTGTTTCGTTGCGCCGTTTTGAATACCAGCCAGACGAG

ACGGGGTTCTACGAATCTTGGTCGATACCAAGCCATTTCCGCTGAATATCGGGGAGCTCACCGCCAGAATCGGTGGT

TGTGGTGATGTACGTGGCGAACTCCGTTGTAGTGCCTGTGGTGGCATCCGTGGCCACTCTCGTTGCACGGTTCGTTG

TGCCGTTACACiGCCCCGTTGACAGCTCACCGA ACGTAGTTA A AACATGCTGGTCA A ACTAGGTTTACCA
ACCiATACG 7470-7546 AGICAGCTCATCTAGGGCCAGITCIAGGCGITGITCGTYGCGCGGTICGTIGCGCATUFLICGIGTGULIGCTAGAT

GGCTCCGCAACCACACGCTTCGAGGTTGAGTGCTTCCAGCACGGGCGCGATCCAGAAGAACTTCGTCGTGCGACTGT

[0170]
Mycobacteria overexpressing disA are attenuated for virulence. As shown in Fig. 12, when mice are infected with 3.5 logio units by the aerosol route of either M.
tuberculosis harboring the pSD5B Phsp60::disA plasmid tb-disA-OE or Mth-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 Phsp6o::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.
[0171] Addition of CDN cyclase genes to rBCG other than disA
[0172] Overexpression of the PAMP immunomodulator, 3'-5' c-di-GMP
by overexpressint 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. 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 4 c-di-GMP + 2 pyrophosphates. The EAL domain (approximately amino acids 350-623) is a diguanylate phosphodiesterase catalyzing the reaction c-di-GMP
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 R507340 protein will produce high levels of c-di-GMP in recombinant BCG.
[0173] SEQ ID NO:4 [0174] 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.

MCNDTATPQLEELVTIVANQLMTVDAATSAEVSQRVLAYLVEQL
GVDVSFLRHNDRDRRATRLVAEWPPRLNIPDPDPLRLIYFADADPVFALCEHAKEPLV
FRPEPATHDYQRLIHEARGVPVTSAAAVPLVSGEITTGLLGFIKFGDRKWHEAELNAL
MTIATLFAQVQARVAAEARLRYLADHDDLTGLHNRRALLQHLDQRLAPGQPGPVAALF
LDLDRLKAINDYLGHAAGDQFIHVFAQRIGDALVGESLIARLGGDEFVLIPASPMSAD
AAQPLAERLRDQLKDHVAIGGEVLTRTVSIGVASGTPGQHTPSDLLRRADQAALAAKH
AGGDSVAIFTADMSVSGELRNDIELHLRRGIESDAL RLVYLPEVDL RTGDIVGTEALV
RWQHPTRGLLAPGCFIPVAESINLAGELDRWVLRRACNEFSEWQSAGLGHDALLRINV
SAGQLVTGGFVDFVADTIGQHGLDASSVCLEITENVVVQDLHTARATLARLKEVGVHI
AIDDFGTGYSAISLLQTLPIDTLKIDKTFVRQLGTNTSDLVIVRGIMTLAEGFQLDVV
AEGVETEAAARILLDQRCYRAQGFLFSRPVPGEAMRHMLSARRLPPTCIPATDPALS
[0175] SEQ ID NO:5 [0176] 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; NCBT Reference Sequence: NC 008769.1; Protein ID WP
003898837.1; old locus tag BCG_1416e; DNA from NC_008769.1:e1548390-1546519 Mycobacterium bovis BCG Pasteur 1173P2). The identical sequence is present in other strains of BCG, e.g., M,vcobacierium 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.
ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGTAGCCAACCAGCTCATGACAG
TCGACGCTGCCACGTCAGCCGAAGTCAGTCAGCGCGTTTTGGCCTATCTAGTGGAACAGCTGGGCGTAGA
TGTCAGCTTTTTGCGTCATAACGATCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATGGCCACCTCGC
CTCAACATACCGGACCCCGATCCGCTCAGGCTGATCTACTTCGCTGATGCCGACCCGGTGTTTGCGCTAT
GCGAACACGCCAAAGAGCCTCTCGTGTTCCGGCCCGAGCCGGCCACCGAGGACTATCAACGCCTCATCGA
AGAAGCCCGCGGGGTTCCGGTAACGTCGGCTGCCGCCGTGCCGCTGGTATCTGGCGAGATCACCACTGGA
CTGCTGGGGTTCATCAAGTTCGGTGATCGGAAATGGCACGAGGCCGAGCTTAACGCCCTCATGACCATCG
CTACACTCTTCGCCCAGGTGCAGGCTCGCGTCGCC GCCGAGGCGCGGCTTCGCTATCTGGCCGACCATGA
CGATCTGACCGGACTCiCA l'AACCGTCGCGCGTIGCTGCAGCACCTGGACCAAAGACTIGGCCCCCGOACAA
CCTGGCCCGGTCGCGGCGCTATTTCTCGACTTGGACCGCCTCAAGGCCATCAACGACTACCTGGGCCACG
CCGCCGGTGACCAGTTCATCCATGTGTTCGCCCAACGGATC GGTGACGCACTCGTTGGCGAGAGCCTGAT
CGCCCGACTCGGCGGCGACGAATTCGTCCTCATACCCGCATCTCCAATGAGTGCCGATGCCGCTCAACCG
CTCGCCGAACGTCTTCGCGACCAGCTCAAGGACCACGTCGCTATCGGCGGTGAGGTGCTCACCCGCACCG
TCAGTATCGGTGTCGCCTCAGGGACTCCCGGACAGCACACAC CGTCGGAC CTCCTGC GCCGAGCCGACCA
AGCCGCTCTGGCAGCCAAACACGCCGGCGGAGATAGCGTCGCGATTTTCACCGCGGACATGTCGGTCAGC
GGCGAACTGCGCAACGATATTGAACIACACCITCGACGI'GGTATCGAATCGGACGCCCATCGCCI'GGTCT
ACCTACCCGAGGTCGACCTACGGACCGGCGACATTGTCGGGACCGAGGCATTGGTCCGGTGGCAGCACCC
CACCCGTGGGCTGCTGGCACCGGGCTGCTTCATCCCTGTGGCCGAATCCATCAACCTTGCAGGCGAATTG
GATAGATOGGTOCTOCGGAGGGCCTOCAATGAATTCTCCGAGIGGCAGTCAGCCOUTITOGGCCACGACG
CGCTGCTGCGTATCAACGTCTCAGCTGGACAGCTGGTGACGGGCGGGTTTGTTGACTTCGTCGCAGACAC
GATCGGCCAGCACGGTCTGGACGCCTCGTCCGTGTGTTTGGAAATCACC GAAAACGTTGTGGTGCAAGAC
CTACATACCGCCAGAGCCACCCTGGCTCGACTCAAAGAAGTCGGCGTTCACATCGCTATCGACGATTTCG
GCACCGGCTATAGCGCCATATCACTGTTGCAGAC GCTACCGATCGACACGCTCAAGATCGACAAAACATT
CGTCiCGGC A ACTCGGA ACC A AC ACTA GCCiA TCTCiGTC ATTGTGCCiCGCiCA TC
ATGACACTCGCCGA A GCiC
TTCCAACTCGATGTAGTAGCCGAAGGCGTCGAGACCGAGGCTGCCGCCAGAATTCTATTGGATCAGCGCT
GTTACCGTGCGCAAG GCTTCTTGTTCTCCCGGCCTGTCCCCGGG GAGGCCATGCGG CACATGTTGTCCGC

ACGACGACTACCGCCGACCTGCATACCTGCAACTGACCCGGCGTTATCTTGA
[0177] SEQ ID NO:6 [0178] 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; NCB1 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.
MCNDTATPQLEELVTTVANQLMTVDAATSAEVSQRVLAYLVEQL
GVDVSF LRHNDRDRRATRLVAE WPPRLNIPDPDPLRLIYEADADPVFALCEHAKEPLV
FRPEPATEDYQRLIEEARGVPVTSAAAVPLVSGEITTGL LGFIKFGDRKWHEAELNAL
MTTATLFAQVQARVA A EA RL RYL A DHDDLTGLHNRR A LLQHLDQRL A PGQPGPVA A LF
LDLDRLKAINDYLGHAAGDQFTHVFAQRIGDALVGESLIARLGGDEFVLIPASPM SAD
AAQPLAERLRDQLKDHVAIGGEVLTRTVSIGVASGTPGQHTP SDLLRRADQAALAAKH
AGGDSVAIFTADMSVSGEL
[0179] SEQ ID NO:7 [0180] 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 1353 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., Mycobacteriutn tuberculosis as a fragment of gene Rv1354c or MT1397, and in Mycobacterium bovis as a fragment of gene Mb1389c.
ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGTAGCCAACCAGCTCATGACAG
TCGACGCTGCCACGTCAGCCGAAGTCAGTCAGCGCGTTTTGGCCTATCTAGTGGAACAGCTGGGCGTAGA
TGTCAGCTTTTTGCGTCATAACGATCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATGGCCACCTCGC
C TCAACATACCGGACCCCGATCCGCTCAGGCTGATCTACTIVGCTGATGCCGACCCGGTUITMCGCTAT
GCGAACACGCCAAAGAGCCTCTCGTGTTCCGGCCCGAGCCGGCCACCGAGGACTATCAACGCCTCATCGA
AGAAGCCCGCGGGGTTCCGGTAACGTCGGCTGCCGCCGTGCCGCTGGTATCTGGCGAGATCACCACTGGA
C IGC1'GGGGITCATCAAGITCGGTGATCGGAAATGGCACGAGGCCGAGCTI'AACGCCCTCATGACCATCG
CTACACTCTTCGCCCAGGTGCAGGCTCGCGTCGCCGCCGAGGCGCGGCTTCGCTATCTGGCCGACCATGA
CGATCTGACCGGACTGCATAACCGTCGCGCGTTGCTGCAGCACCTGGACCAAAGACTGGCCCCCGGACAA
CCIGGCCCOGTCGCGGCOCIAI"ITCTCGACTIGOACCGCCTCAAGGCCAICAACGACTACCTOGCiCCACG
CCGCCGGTGACCAGTTCATCCATGTGTTCGCCCAACGGATC GGTGACGCACTCGTTGGCGAGAGCCTGAT
MCCCGACTCGGCGGCGACGA ATTMTCCTCA TA CCCGC ATCTCC A A TGA GTGCCGA TGCCGCTCA ACCG

CTCGCCGAACGTCTTCGCGACCAGCTCAAGGACCACGTCGCTATCGGCGGTGAGGTGCTCACCCGCACCG
TCAGTATCGGTGTCGCCTCAGGGACTCCCGGACAGCACACACCGTCGGACCTCCTGCGCCGAGCCGACCA
AGCCGCTCTGGCAGCCAAACACGCCGGCGGAGATAGCGTCGCGATTTTCACCGCGGACATGTCGGTCAGC
GGCGAACTG
[0181] Overexpression of the PAMP immunomodulator, 2'-5'c-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'-5'c-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'-5'c-GAMP in recombinant BCG.
[0182] SEQ ID NO:8 [0183] Cyclic GMP-AMP synthase, DncV, from Vibrio cholerae, amino acid sequence (436 amino acids; UniProtKB/Swiss-Prot Protein ID Q9KVG7.1).
MRMTWNFHQYYTNRNDGLMGKLVLTDEEKNNLKALRKIIRLRTRDVFEEAKGIAKAVKKSALTFEIIQEK
VSTTQIKHL SD SEQREVAKLIYEMDDDARDEFLGLTP RFWTQ GSFQYDTLNRPFQPGQEMDIDDGTYMPM
PIFESEPKIGHSLLILLVDASLKSLVAENHGWKFEAKQTCGRIKIEAEKTHIDVPMYAIPKDEFQKKQIA
LE ANR SFVKGA TFESYVA DSITDDSETYE LDSENVNL A LR EGDR K WINS DPKTVEDWFND SCTR
TGKHLR
KVC RFMKAWRDAQWDVGGPS SI S LMAATVNILDSVAHDASDLGETMKIIAKHLP SEFARGVESP DSTDEK
PLFPPSYKHGPREMDIMSKLERLPEIL SSAESADSKSEALKKINMAFGNRVTNSELIVLAKALPAFAQEP
SSASKPEKISSTMVSG
[0184] SEQ ID NO: 9 [0185] Cyclic GMP-AMP synthase, DncV, from Vibrio cholerae, DNA
sequence (1311 nucleotides [436 eodons 1 stop codon]; encodes UniProtKB/Swiss-Prot Protein ID
Q9KVG7.1;
NCBI Reference Sequence: NC 002505.1: Vibrio cholerae 01 biovar El Tor str.

chromosome I, complete sequence, and nucleotides 180419-181729) Ci IGAGAA TGAC 1TGGAAC f 1TCACCAGIAC 1ACACAAACCGAAA TGAIGGC 1TGA IGGGCAAGC lAG
ITC
TTACAGACGAGGAGAAGAACAATCTAAAGGCATTGCGTAAGATCATCCGCTTAAGAACACGAGATGTATT
TGAAGAAGCTAAGGGTATTGCCAAGGCTGTGAAAAAAAGT GCTCTTACGTTTGAAATTATTCAGGAAAAG
G IGICAACGACCCAAAITAAGCACC ITTC 1 GACAGCGAACAACGAGAAG IGGrC TAAGC 1TA 1TACGAGA

TGGATGATGATGCTCGTGATGAGTTTTTGGGATTGACACCTCGCTTTTGGACTCAGGGAAGCTTTCAGTA
TGACACGCTGAATCGCCCGTTTCAGCCTGGTCAAGAAATGGATATTGATGATGGAACCTATATGCCAATG
CCTATTTTTGAGTCAGACiCCTAAGATTCiGTCATTCTTTACTAATTCTTCTTCiTTCiACCiCCiTCACTTAAGT
CACTTGTAGCTGAAAATCATGGCTGGAAATTTGAAGCTAAGCAGACTTGTGGGAGGATTAAGATTGAGGC
AGAGAAA ACACATATTGATGTACCAATGTATGCAATCCCTAA AGATGAGTTCCAGA AA AAGCA A ATAGCT

TTAGAAGCAAATAGATCATTTGTTAAAGGTGCCATTITTGAATCATATGTTGCAGATTCAATTACTGACG
ATAGTGAAACTTATGAATTAGATTCAGAAAACGTAAACCTTGCTCTTCGTGAAGGTGATCGGAAGTGGAT
CAATAGCGACCCCAAAATAGTTGAAGATTGGTTCAACGATAGTTGTATACGTATTGGTAAACATCTTCGT
AAGGTTTGTCGCTTTATGAAAGCGTGGAGAGATGCGCAGTGGGATGTTGGAGGTCCGTCATCGATTAGTC
TTATGGCTGCAACGGTAAATATTCTTGATAGCGTTGCTCATGATGCTAGTGATCTCGGAGAAACAATGAA
GATAATTGCTAAGCATTTACCTAGTGAGTTTGCTAGGGGAGTAGAGAGCCCTGACAGTACCGATGAAAAG
CCACTCTTCCCACCCTCTTATAAGCATGGCCCTCGGGAGATGGACATTATGAGCAAACTAGAGCGTTTGC
CAGAGATTCTGTCATCTGCTGAGTCAGCTGACTCTAAGTCAGAGGCCTTGAAAAAGATTAATATGGCGTT
TGGGAATCGTGTTACTAATAGCGAGCTTATTGTTTTGGCAAAGGCTTTACCGGCTTTCGCTCAAGAACCT
AGTICAGCCTCGAAACCTGAAAAAATCAGCAGCACAATGGTAAGTGGCTGA
[0186] 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'-3'c-GAMP in recombinant BCG.
[0187] SEQ ID NO:10 [0188] Cyclic 2'3'-GMP-AMP synthase, cGAS, from Homo sapiens, amino acid sequence (522 amino acids, UniProtKB/Swiss-Prot Protein ID Q81\1884.2).
MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKEGPARKSGSRQKKSAPDT
QE
REPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRACiSCRQRGARCSTKPRPPPGPWD
VP SPG L PVSAPILVRRDAAPG ASKL RAVLEKLKL SRDDISTAAG MVKGVVDHLLLRLKCDSAFRGVG LLN

TGSYYEHVKISAPNEFDVMFKLE VPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEIL SASKMLSKF
RKIIKEEINDIKDTDVIMKRKRGG SPAVTLLISEKISVDITLALE SKS SWPASTQEGLRIQNWL SAKVRK
QLRL KPFYLVPKHAKEGNGFQEETWRL SF SHIEKEILNNHGKSKTCCENKEEKCC RKDCLKLMKYLLEQL
KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFL QCLRTEKLENYFIPEFNL
FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF
[0189] SEQ ID NO: 11 [0190] Cyclic 2'3'-GMP-AMP synthasc, 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 nucleotides [522 codons, 1 stop codon], start codon ATG [bold underlined] at nucleotide 140; Stop codon TGA (bold, underlined) at nucleotide 1706]).
AGCCTGGGGTTCCCCTTCGGGTCGCAGACTCTTGTGTGCCCGCCAGTAGTGCTTGGTTTCCAACAGCTGC
TGC TGGC TCTTCCTCTTGCGGCCTTTTCCTGAAAC GGATTCTTCTTTCGGGGAACAGAAAGCGCCAGCCA
TGCAGCCTTGGCACGGAAAGGCCATGCAGAGAGCTTCCGAGGCC GGAGCCACTGCCCCCAAGGCTTCCGC
ACGGAATGCCAGOGGCGCCCCGATOGATCCCACCGAGTC ---------ICCGGCTGCCCCCGAGGCCGCCCTGCCIAAG
GCGGGAAAGTTCGGCCCCGCCAGGAAGTCGGGATCCCGGCAGAAAAAGAGCGCCCCGGACACCCAGGAGA
GGCCGCCCGTCCGCGCAACTGGGGCCC GCGC CAAAAAGGCCCCTCAGCGCGCCCAGGACACGCAGCCGTC
TGACGCCACCAGCGCCCCIGGGGCAGAGGGGCTGGAGCCTCCTGCGGCTCGGGAGCCGGCTCTTTCCAGG
GCTGGTTCTTGCCGCCAGAGGGGCGCGCGCTGCTCCAC GAAGCCAAGACCTCCGCCCGGGCCCTGGGACG
TGCCCAGCCCCGGCCTGCCGGTCTCGGCCCCCATTCTCGTACGGAGGGATGCGGCGCCTGGGGCCTCGAA
GCTCCUGGCGGTTTTGGAGAAGTTGAAGCTCAGCCGCGATGATATCTCCACGGCGGCGGGGATGOTGAAA
GGGGTTGTGGACCACCTGCTGCTCAGACTGAAGTGCGACTCCGCGTTCAGAGGCGTCGGGCTGCTGAACA
CC GGGA CiC TA CT A TGA GC A CGTGA A GA TTTC TGC A CC T A A TG A A TTTGA TGTC
ATGTTT A A A CTGGA A GT
CCCCAGAATTCAACTAGAAGAATATTCCAACACTCGTGCATATTACTTTGTGAAATTTAAAAGAAATCCG
AAAGAAAATCCTCTGAGTCAGTTTTTAGAAGGTGAAATATTATCAGCTTCTAAGATGCTGTCAAAGTTTA
GGAAAATCATTAAGGAAGAAATTAACGACATTAAAGATACAGATGTCATCATGAAGAGGAAAAGAGGAGG
GAGCCCTGCTGTAACACTTCTTATTAGTGAAAAAATATCTGTGGATATAACCCTGGCTTTGGAATCAAAA
AGTAGCTGGCCTGCTAGCACCCAAGAAGGCCTGCGCATTCAAAACTGGCTTTCAGCAAAAGTTAGGAAGC
AACTACGACTAAAGCCATTTTACCTTGTACCCAAGCATGCAAAGGAAGGAAATGGTTTCCAAGAAGAAAC
ATGGCGGCTATCCTTCTCTCACATCGAAAAGGAAATTTTGAACAATCATGGAAAATCTAAAACGTGCTGT
GAAAACAAAGAAGAGAAATGTTGCAGGAAAGATTGTTTAAAACTAATGAAATACCTTTTAGAACAGCTGA
AAGAAAGGTTTAAAGACAAAAAACATCTGGATAAATTCTCTTCTTATCATGTGAAAACTGCCTTCTTTCA
CGTATGTACCCAGAACCCTCAAGACAGTCAGTGGGACCGCAAAGACCTGGGCCTCTGCTTTGATAACTGC
GTGACATACTTTCTTCAGTGCCTCAGGACAGAAAAACTTGAGAATTATTTTATTCCTGAATTCAATCTAT
TCTCTAGCAACTTAATTGACAAAAGAAGTAAGGAATTTCTGACAAAGCAAATTGAATATGAAAGAAACAA
TGAGTTTCCAGTTTTTGATGAATITTGAGATTGTATTTTTAGAAAGATCTAAGAACTAGAGTCACCCTAA
ATCCTGGA GA ATAC A A GA A A A ATTTGA A A A GGGGCCA GACGCTGTGGC TCAC
[0191] SEQ ID NO: 12 [0192] Cyclic 2'3'-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).
A TCiCA A CCATGGCACCiGG A A A GCC A TG C A GCGT CiCCi A GCCiA ACiCCGGGGCG A
CGG CCCCC A A GGCGTCGGCGCGT A A CGCGCG
GGG TGC GCCCATGGACCCGACCiGAGTCCCCCGCGGCGCCGGAGGC GGCCCTCiCCGAAAGCGGGTAAGTTCGG
TCCAGCCiCGGA
AAAGCGGGAGCCGCCAAAAGAAGICCGCGCCCGACACCCAGGAGCGICCCCCGGICCGGGCCACCGGCGCGCGIGCCAA
AAAA
GCCCCGCAACGGGCGCAAGATACGCAGCCAAGCGATGCGACCTCCGCCCCCGGGGCGGAGGGTCTGGAGCCCCCGGCCG
CCCG
GGAGCCAGC GCTCTCGCGCGCGGGTTCCTGCCGTCAGCGGGGCG
CGCGGTGTTCCACGAAACCCCGTCCCCCACCAGGTCCCT
GGGACGTGCCGTCGCCGGGTTTGCCGGTGAGCGCGCCAATCCTGGTCCGGCGCGACGCGGCCCCGGGGGCGTCGAAATT
GCGT
GCGGTGCTCGAGAAATTGAAGTTGTCGCGCGACGACATCTCCACGGCCGOGGGTATGGTCAAGGGCGTGGTCGATCATT
TGTT
GTTGCGGCTCAAGTGTGATTCGGCGTTCCGCGGGGTGGGCTTGCTGAACACG
GGGTCCTACTATGAGCATGTCAAAATCAGCG
CCCCCAACGAATITGACGIGATGITTAAGCTUGAAGIGCCACGTATCCAATIGGAAGAGIATICCAATACCCGIGCGTA
TTAT
TTCGTCAAATTTAACiCGCAATCCCiAACiGAAAATCCACTCAGCCAATTCTIGGAGOGCGAAATTCTGICGGCCTCGA
AAATGCT
CTCCAAATTTCGTAAGATTATCAAGGAGGAGATCAACGACATTAAGGACACGGATGTGATCATGAAACGTAAACGTGGC
GGTT
CCCCCGCGGTGACGCTCCTCATTTCGGAAAAAATTTCGGTGGACATTACCCTGGCGTIGGAATCGAAGTCCAGCTGGCC
GGCG
TCCi A CCC A CiciA CiCiCiCCTGCCiCi A TTCA A AA CTC1CiTTGA GCGCCA AA CiTC1CCA1A
MiCACi CTGCciTCTCA A A CCCTTTTA TT TCA1TC
CCGAAACATGCCAAAGAGGGTAACGGTTTTCAAGAGGAAACCTGGCGTTTGAGCTTCTCCCACATTGAGAAGGAGATTT
TGAAC
AACCATtiGTAAGTCCAAAACCiTGCTGCGAGAATAAGCiAAGAAAAATGTIGTCGCAAAGATTGICICAAATTGATGA
AATAITTG
CTGGAACAACTCAAAGAGCGTTTTAAGGACAAGAAGCATCTCGACAAGTTCTCCTCGTATCACGTCAAGACCGCCT
TCTTTCAT
GTCTGTACGCAGAACCCGCAAGATAGCCAGTGGGATCGCAAGGACTTGGGGTTGTGTTTTGACAATTGCGTCACCTATT
TCTTG
CAATGTTTGCGGACCGAGAAATTGGAGAACTACTT
TATTCCAGAATTCAACTTGTTTTCCTCGAATCTGATTGACAAACG CTCC
AAAGAGTTTCTGACGAAGCAGATTGAATACGAGCGTAACAATGAGTTTCCGGTCTTTGACGAGTTTTGA
[0193] SEQ ID NO:13 [0194] Plasmid pMH94H-Phsp6o::disA::hcGASco::mCherry which is an E. co/i-mycobacterial shuttle plasmid that overexpresses the BCG disA gene, the human cGAS gene (with mycobacterial codon optimization), and mCherry from the Pl in troduced into promoter, DNA sequence.
When into BUG, M. tuberculosis, M. bovis or highly related strains, this plasmid integrates as a single copy in the mycobacterial chromosome (10842 nucleotides; promoter Ph,p6o DNA
comprised of a portion of the M. leproe 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).
TCGCGCOTTTCGGTGATGACCGTGAAAACCICTGACACATGCAGCTCCCGGAGACCGTCACACCTIGTCTGTAAGCGGA

GGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGC

TGTACTGAGAGTGCACCAAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTA A A

AACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTT

ACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACG

CC A TC A CCC A A A TC A A GTTTTTTCGGGTC GGT GCC GT A A A GC A CT A A A TCGG A

TTGACCGGGAAAGCCGGCGAACCTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGACCGGGCGCTAGGGCGCTGGCAAGT

CGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCOTACTATGGTTGCTTTGACGT

TGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGG

GCGATCGGTGCGGGCCTCTTCGCT ATT ACGCCAGCT GGCGAAAGGGGGATGT GCT GC A GGCGATT

GGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAAATTCCGG

GCAC TCGCCTTAGGGGAGTGC TAAAAATGATCCTGGCACTC GC GATCACC GAGT GCCAGGTC GG

CAACCTGTGGTCGTCCGTCCCGGGCACTCCACCCGGCCAGCGTAAGTAATGGGCGTTGTCCGCACCCGCTGACATGCAC

TGACTCGTCC GACC CT GCGTGAGGC TGTCGCCC GCCTAGCC CC GGGCAC TGGGC TGC GGGAC

GGCCGCACTGGTGCCCTGATCGTGCTGGGCCATGACGAGAATGTCGAGGCCATCTGCGATGGTGGCTTCTCCCTCGATG

CT ATGCAGCAACCCGGCT

CCAACGTGCAACTGGTACCGGATCCGTCGATCCCCACCGACGAATCGGGGACCCGCCACCGCTCGGCCGAGCGGGCCGC

CAGACCGGTTACCCGCTGATCTCAGTGAGCCACTCGATGAACATCGTGACCGTCTACGTCCGCCGGGAACCTCACGTAT

CGACTCCGCAACCATCCTGTCGCGCGCCAACCAGGCCATCGCAACCCTCGAGCCGTACAAAACCAGGCTCGACGAGGTC

GGCAACTGTCCAGGGCAGAAATCGAGGACTTCGTCACGCTGCGCGATGTGATGACCGTGGTGCAACGCCTCGAGCTGGT

CGAATCGGGCTGGTGATCGACTACGACGTGGTC

CGGCGGCAACGAC ACC GCC CGGGAATT G AT C GT GCGCG AT T ACCACGCC

C:CAC:CCTGOACCiA ACTOCrAC'OCC:C: R4 (1CA COG( 'GACC ITT ' ICOA: IT
ICACCGCGC:TOGC AA AGGIIIIVGGAT A IrCGACG 1826-1908 ACCACGGAAGCGCAGGATTCGACGCTGAGCCCGCGTGGCTACCGCGCGATGGCCGGTATCCCCCGGCTCCAGTTCGCCC

CGACCTGCTGGTCCGGGCGTTCGGAACGTTGCAGGrGTCTGCTGGCGGCCAGCCCCGGCGATCTGCAATCAGTGGACGG

GC GC CATGT GGGCC C GTCATGTGCGC GAGGGGT TGTCACAGC TGGC GGAATC GACCATCAGC

ATGCAACCATGGCACGGGAAAGCCATGCAGC GT GC GAGCGAAGCC GGGGCGACGGC CCCCAAGGC GTC GGC

GGGTGCGCCCATGGACCCGAC7GGAGTCCCCCGC7GGCGCCGGAGGCGGCCCTGCCGAAACiCGGGTAAGTTCGCTCCA

AAAGCGGGAGCCGCCAAAAGAAGTCCGCGCCCGACACCCAGGAGCGTCCCCCGGTCCGGGCCACCGGCGCGCGTGCCAA

GCCCCCiCA A CGOGCGCA AGA TA CGC A GCCA A GCGA TGCGA CC TCCGCCCCCGGGGCGG A
GGOTCTOCrA CiCCCCCGGCCGCCCCr 2407-2489 GGAGCCAGCGCTCTCGCGCGCCiGGTTCCTGCCGTCAGCGGGGCGCGCGOTOTTCCACGAAACCCCGTCCCCCACCAGG

GGGACGTGCCGTCGCCGGGTTTGCCGGTGAGCGCGCCAATCCTGGTCCGGCGCGACGCGGCCCCGGGGGCGTCGAA_AT

ITiCOCCTCA A (i 1(1 INA It :GOCG FCCOCOGOOTOGGC OCIOA A C A :(1(10GTCC: IA (7 IA1 GA GC A 1:(1 IVA A AA IC AOCCi 2739-2821 CCCCCAACGAATTTGACGTGATGTTTAAGCTGGAAGTGCCAC

TTCGTCAAATTTAAGCGCAATCCGAAGGAAAATCCACTCAGCCAATTCTTGGAGGGCGAAATTCTGTCGGCCTCGAAAA

CTCCAAATTTCGTAAGATTATCAAGGAGGAGATCAACGACATTAAGGACACGGATGTGATCATGAAACGTAAACGTGGC

CCCCCGCGGTGACGCTCCTCATTTCGGAAAAAATTTCGGTGGACATTACCCTGGCGTTGGAATCGAAGTCCAGCTGGCC

TCGACCCAGGAGGGCCTGCGGATTCAAAACTGGTTGAGCGCCAAAGTGCGGAAGCAGCTGCGTCTCAAACCCTTTTATT

CCOAAACA FOCCAAAOACOU TAACCUFT1 TCAAGAGOAAACC 1tiOCCi1116ACC1IC

AACCATGGTAAGTCCAAAACGTGCTGCGAGAATAAGGAAGAAAAATGTIGTCGCAAAGATTGTCTCAAATTGATGAAAT

CTGGAACAACTCAAAGAGCGTTTTAAGGACAAGAAGCATCTCGACAAGTTCTCCTCGTATCACGTCAAGACCGCCTTCT

GT CTGT ACGC A GA ACCCGC A A GAT AGC CA GTGGGA TCGCA A GG ACTTGGGGTT GTGTTTTGA

CAATGTTTGCGGACCGAGAAATTGGAGAACTACTTTATTCCAGAATTCAACTTGITTTCCTCGAATCTGATTGACAAAC

AAAGAGTTTCTGACGAAGCAGATTG A A

AGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGACTICATGCGCTTCAAGGIGCACATGGAGGGCTCCGTGAACG
(3CCAC 3742-3825 GAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTG

CTGCCCTTCGCCTGG CACATCCTCTCCCCTCAGTTCATCTACCG CTCCAAGC CCTACCTCAAGCACCCCC CC

T ACTTGAAGCT GTCCTTCCCCGAGGGCTTCAAGT GGGAGCGC GT GATGAACTTCGAGGACGGCGGCG

GACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAA

A AGA AGA( :( :A I CGGC ITiGGAGGC( T:1:( :( AGCGGA IA( :( :( :GAGGACGOCGC( :( !CAA GGG( :GAGA: IC A AG( :A GA GGCTG 4162-4225 AAGCTGAAGGACCGCGGCCACTAC

AACGTCAACATCAAGTTGGACATCACCTCCCACAACGAOGACTACACCATCGTGGAACAGTACOAACGCGCCGAGGGCC

TCCACCGGCGGCATGGACGAGCTGTACAAGTAGACTAGTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCAT

ACTCAGAAGTGAAACGCGGT AGCGCCGATGG TACT GT G GGGT CTCCCCAT GC

CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGT TT

GGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAA

GCAGAAGGCCATCCTGACGGATGGCCTTTTTTCTAGAGTCGACCACCAAGGGCACCATCTCTGCTTGGGCCACCCCGTT

CAGCCAGCTCGCTGAGAGCCGTGAACGACAGGGCGAACGCCAGCCCGCCGACCIGCCIAGGGTTCCGACCGCTGCAACT

CAACCTTGTCCCGGTCTATTCTCTICACTGCACCAGCTCCAATCTGGTGTGAATGCCCCTCGTCTGTTCGCGCAGGCGG

TCTATTCGTTTGTCAGCATCGAAAGTAGCCAGATCAGGGATGCGTTGCAACCGCGTATGCCCAGGTCAGAAGAGTCGCA

GT TGCA GACCCCTGGA A AGA A AAA TGGCCAGAGGGCGA AA A CA CCCTCTGACCA GCGGA

TAGCTAGTTTGGAAGAATGGGIGICTGCCGACCACATATGGGCCGGTCAAGATAGGTTITTACCCCCTCTCGGCTGCAT

AAGTGGAAAGAAATTGCAGGTCGTAGAAGCGCGTTGAAGCCTGAGAGTTGCACAGGAGTTGCAACCCGGTAGCCTTGTT

CGAGAGGAGACCTAGTTGGCACGTCGCGGATGGCGATCGCTGAAGACTCAGCGCAGCGGGAGGATCCAAGCCTCATACG

CCGCAGGACGGTGTGAGGTACTACGCGCTGCAGACCTACGACAACAAGATGGACGCCGAAGCCTGGCTCGCGGGCGAGA

CTCATCGAGATGGAGACCTGGACCCCTCCACAGGACCGGGCGAAGAAGGCAGCCGCCAGCGCCATCACGCTGGAGGAGT

CGGA A G
' KiCiA GC:GC:GMT'. !MC A GACCiGC A CC A CiGGA.I'r l'A CACCGGGC A CcieGC4A
GCGCCGC A. I I' A CCCGG 5590-5673 CTAGGTGAAGTGGCGGTCACAGAGATGACGCCAGCTCTGGTGCGTGCGTGGTGGGCCGGGATGGGTAGGAAGCACCCGA

CGCCGGCATGCCTACAACGTCCTCCCiGGCGGTGATGAACACAGCGGTCGAGGACAAGCTGATCGCAGAGAACCCGTGC

GAGCAGAAGGCAGCCGATGAGCGCGACGTAGAGGCGCTGACGCCTGAGGAGCTGGACATCGTCGCCGCTGAGATCTTCG

TACCGGATCGCGGCATACATCCIGGCGTGGACGAGCCICC

GACGGCATGACGATGAAGCTCCGGGTGCGCCGTGGCGCTTCCCGCGTGGGGAACAAGATCGTCGTTGGCAACGCCAAGA

CGGTCGAAGCGTCCIGTGACGGTTCCGCCTCACGTCGCGGAGATGATCCGAGCGCACATGAAGGACCGTACGAAGATGA

GOCCCC GA CrGC ATT CCT G ACC ACCIA CGC AGGGC A A CCGGCT GT C GA A

GCCAAGATCGGTCGGCCGGAACTCCGCATCCACGACCTCCGCGCTGTCGGCGCTACGTTCGCCGCTCAGGCAGGTGCGA

AAGGAGCTGATGGCCCGTCTCGGTCACACGACTCCTAGGATGGCGATGAAGTACCAGATGGCGTCTGAGGCCCGCGACG

ATCGCTGAGGCGATGTCCAAGCTGGCCAAGACCTCCTGAAACGCAAAAAGCCCCCCTCCCAAGGACACTGAGTCCTAAA

(ierei FIR:FIVI( :A ( IA( :OCCiA AGA ACCACOCCTOGCCOC :(iA :( AOC ACCOCC:0( :1(71(i FOCOGAGAC:C IGOOCACCAOCCC 6514-6597 CGCCGCCGCCAGGAGCATTGCCGTTCCCGCCAGCTGAGTTCTGTTGTGCGCCGCCTATGTAGAGCTGGTCGTTGTAGGT

TCT CCAGGCG ACTTTCC GGCG AC GCTG AGGATGTC GAT C AC AGAGCC TCCGGGAC CGC C GGTT

GACAGCGGACGCCGTGGTGTTTCCTCCAGGGCCTCCGGCCTIGCCTGAGAATACAGAGCCAGCTCCCGCTGCGCCTCCA

CGACGAGCCCGGTGATCGTCTTGGTCGACCTGCAGGCATGCAAAAGCTGATCCTTGCCGAGCTGGGATGGAAGCCCGGC

CCACCCTGGAGGAGAT GATC G AGGATGC C AGGGC CTTT C ACGCCC GCC GC T GCT GAGCGT CC

FCGGCCOUCCCGC FCCGOCiC fCCiCAGCAGCGGGC fCGOCUCGGOCCCGGGCiC
ICCCCGOCCOCCOGOCCiCiGGCFCCGCCCGG 7013-7095 CGGCCGCCOGGOGCCGGGGGCGGCGCCGGGCGOCCCGOGGCGTCAGGCGCCGGGGGCGGTGTCCGGCGGCCCCCAGAGG

GCGCCAGTTCCTCCGGATCGGTGAAGCCGGAGAGATCCAGCGGGGTCTCCTCGAACACCTCGAAGTCGTGCAGGAAGGT

GCGAGCAGTTCGCGGGCGAAGTCCTCGGTCCGCTTCCACTGC GCCCCGTCGAGCAGCGCGGCCAGGATCTCGC

GAAGGCGTTGAGATGCAGTTGCACCAGGCTGTAGCGGGAGTCTCCCGCATAGACGICGGTGAAGTCGACGATCCCGGTG

CGGTCGCGGCCAGGTCCACGAAGATGTTGGTCC CGTGCAGGTCGCCGTGGAC GAACCGGGGTTC

TCCACGTCC GGCAGCCAGTCCTC CAGGCGGTCCAGCAGCC GGGGC GAGAG GTAGC CC CACCC GC GGT

CGC GCGGC GTTCCCGCAGCAGTTCC GGGAAGAC CTCGOAAT GGGGGGTGAGCAC G GT GTTCC

GCCGCCCGAGCACCCGGCCGAGTTCGCGG GCCAC CGAGCAGC GC G T TC CGGTC G G TC G TGCC

GT GGTGCCGGTCATCCGGCTC AT CACCAGGT

GCGGGGCACCGGC ACC GGGGCGTC CGCCAGGACCGCGT ACGC CT CC GAC T CCGAC GCGAGGCTC TCC

CGCCGAACAGC f [GA! CACCGGGC CGGGC IC GC C GACCA6 fACGGGCi f _EGG TOCIC f ACCGGCAGCCCGAGCTCCTCCAGGGCTCGGCGGGCCAGCGGCTCCCAGAATTCCTGGTCGTTCCGCAGGCTCGCGTAGG

ATCCGAAICAA fACOG ICGAGAAG fAACAGO GA f IC! ICACAGC GOACCIC lA f ICACAGGG
IACGCOCCGOCY f 1092-8174 CCGCACGGCCGGTCGCGACACGGCCTGTCCGCACCGCGGATCAGGCGTTGACGATGACGGGCTGGTCGGCCACGTCGGG

TTCTCGGTGGTGCTGCGGTCGGGATCGCCAATCTCTACGGGCCGACCGAGGCGACGGTGTACGCCACCGCCTGGTTCTG

GGCG A GGCGCCGTCCC A GGCCCC.GCC GA TCCCC GTCCCCC GC GTCGTCG A GCGCGGT GCCG
AC.G AC A CC GCCCrCG TGGC TCGT 8341-8473 CACGGAGGCCGTCCCCGGCGTCGCGGCGGCCGAGGAGTGGCCCGAGCACCAGCGGITCGCCGTGGTCGAGGCGATGGCG

TGGCCCGCGCCCTCCACGAGCTGCCCGTGGAGGACTGCCCCTTCGACCGGCGCCTCGACGCGGCGGTCGCCGAGGCCCG

AACGTCGCCGAGGGCCTGTGGACCTCCACGACCTGCAG

TACGAGCCGGAAGCATAANGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT

GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG

GEC r CGC [ fCC1CGC [CAC FGAC fCGC fGCGGIVGG fCG GGC IGCGGCGAGCGCiTAtCAGC [CAC

TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA

GCCGCGTTGCTGViCGITTTTCCATAGGCTCCCiCCCCCCTGACC_TAGCATCACAAAAATCGACGCTCAAGTCAGAGG

CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCIGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTT

GATACCIGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGIATCTCAGTTCGGTGTA

GT TCGCTCCAAGCT GGGCTGT GTGCACGAACCCCCCGTTCAGCC CGACCGCTGCGC

CAACCCGGTAAGACACGAC

CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT

TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC

TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA

AAGGGATTTTGGTCATGAGAT TATCAAAAAGGATCTTCACCTAGATCCT TT

ATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG

CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC

ACTTTATC CGCCT C CATCCAGTC TATTAATTGTTGCC GGGAAGC

GT TGCCATTGC TACAG GCATC GTGGTGTCACGCTC GTC GTTTGGTATGGC TTCAT TCAGC TCCGGTTC

GT TACATGATCC CCC AT GTTGTGCAAAAAAGC GGT TAGC TCCTTC GGTCCTC

l'A IV AC ICA MG l'A IGGCA(TCAC IGCA IAA I IC FC !AC 1G ICA IGCCA I CCG IA AGA
INC IICIG !GAL IGGIGAGIAC 10254-10337 TCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGCTCGTCAATACGGGATAATACCGCG

AGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGAT

TC GATGTAAC CC AC TC GTGCAC C CAAC TGATCTTCAGCATC TTTTAC TTTCACCAGC GTTTC

CAAAATGC C GCAAAAAAGGGAATAAGGGC GACAC GGAAATGTTGAATACTCATACTCTTCC TTT TT

TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT A A A CAAAT_AGGGGT T

CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT

[0195] Knocking out endogenous BCG phosphodiesterase genes and intragenic segments encoding phosphodiesterase domains in order to increase CDN PAMP and DAMP
levels [0196] Overexpression of CDNs by knocking out an endogenous BCG
phosphodiesterase:

[0197] The BCG AHM08589.1 protein encodes a 316 amino acid endogenous bifunctional c-di-AMP and eGAMP phosphodiesterase in BCG that is 100% identical to the Al 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'-S' c-di-AMP (bacterial PAMP molecule) and 2' -3'cGAMP
(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' -3'cGAMP) in recombinant BCG.
[0198] SEQ ID NO: 14 [0199] 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.
MDAVGAAALLSAAARVGVVCHVHPDADTIGAGLALALVLDGCGKRVEVSFAAPATLPESLRSLPGCHLL
VRPEVMRRDVDLVVTVDIPSVDRLGALGDLTDSGRELLVIDHHASNDLFGTANFIDPSADSTTTMVAEILD
AWGKPIDPRVAHCIYAGLATDTCiSFRWASVRGYRLAARLVEIGVDNATVSRTLMDSHPFTWLPLLSRVLG
SAQLVSEAVGGRGLVYVVVDNREWVAARSEEVESIVDIVRTTQQAEVAAVFKEVEPHRWSVSMRAKTVN
LAAVASGFGGGGHRLAAGYTTTGSIDDAVASLRAALG
[0200] SEQ ID NO: 15 [0201] 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 Ry2837c or MT2903, and in Mycobacterium hovis encoding protein Mb2 g 62e.
GTGGACGC CGTCGGTGCCGCTGCGCTGTTGTC GGCCGC TGCCAGGGTCGGGGTAGTCTGCCACGTC CA
CCCCGATGCCGACACCATCGGC GCCGGATTGGCATTGGCATTGGTGTTGGACGGGTGCGGCAAGCGGG
TAGAGGTCAGCTTTGCC GCGCCGGCGACACTGCCCGAGTCGCTGCGTTC GCTGCCGGGCTGC CATC TG
CTGGTCCGCCCTGAGGTGATGCGCCGCGATGTCGATTTGGTTGTGACTGTTGACATTCCGAGTGTTGAT
CGGCTCGGTGCTCTGGGCGATCTAACTGATTCCGGGCGGGAGCTCCTGGTAATCGACCATCACGCCTC
CAACGACCTGTTCGGCACCGCGAATTTCATTGACCCGTCGGCGGATTCCACCACGACGATGGTTGCCG
AGA TCCTCGACGCGTG GGGGA A ACCGA TAGACCCGCGCGTCGCGC A CTGC A TCTACGC CGGGTTGGCG

ACCGACACGGGGTCGTTTCGCTGGGCCAGTGTGCGGGGGTATC GGCTGGCGGCGCGGCTGGTAGAGAT
CGGTGTGGACAACGCCACCGTCAGCAGGACCTTGATGGACAGCCATC CCTTCACCTGGTTGCCGTTGC
TATC GC GGGTGTTGGGTTCGGCGCAGC TGGIGTCC GAGGCGGTC GGTGGCCGCGGGCTGGTTTACGTC
GTCGTCGACAACCGGGAGTGGGTCGCTGCGCGCTCGGAGGAAGTGGAAAGCATCGTCGACATCGTCC
GCACCACGCAACAAGCCGAGGTCGCGGCGGTGITCAAGGAGGTCGAACCGCATCGGTGGTCGGTGTC
GATGCGGGCTAAGACCGTGAATTTGGC CGCGGTTGCCTC TGGGTTCGGTGGCGGTGGTCAC C GGCTGG
CCGCGGGGTATACGACCACCGGCTCGATCGACGACGCTGTGGCGTCGTTGCGCGCGGCGCTTGGTTAG
[0202] SEQ ID NO: 16 [0203] Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP
(also called CnpB, Ry2837c, 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; NCB I/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.
MTTIDPRSELVDGRRRAGARVDAVGAAALLSAAARVGVVCHVHPDADTIGAGLALALVLD
GCGKRVEVSFAAPATLPESLRSLPGCHLLVRPEVMRRDVDLVVTVDIPSVDRLGALGDLT
DS GRELLVIDHHASNDLF GTANFIDP SAD STTTMVAEILDAWGKPIDPRVAHCIYAGLAT
DTGSFR WA SVRGYRL A ARLVETGVDNATVS RTLMDSHPFTWLPLL SRVL GSA QLVSEAVG
GRGLVYVVVDNREWVAARS EEVESIVDIVRTTQQAEVAAVFKEVEPHRWSVSMRAKTVNL
AAVASGFGGGGHRLAAGYTTTGSIDDAVASLRAALG
[0204] Overexpression of CDNs by knocking out an endogenous BCG
phosphodiesterase domain: EAL domain of protein BCG R507340 (previously BCG 1416c). The BCG

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 Ry1354c 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. The GAF domain (approximately amino acids 1-190) is a regulatory domain which influences the activity of the other domains. The GGDFF domain (approximately amino acids 190-350) is a diguanylate cyclase catalyzing the reaction 2 GTP 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
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.
[0205] 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 /1//. tuberculosis Rv1357c protein over its C-terminal 287 amino acids. The domain structure of BCG AHM07112 is that of a single EAL domain. As the M.
tuberculosis Rv1357c protein is known to cleave 3'-S' 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.
[0206] SEQ ID NO: 17 [0207] 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 diguanylatc phosphodiesterasc of Mycobacterium tuberculosis protein Rv1357c or MT1400 and of Mycobacterium bovis as protein Mb1392c.
MID YEEMFRGAMQARAMVAN PDQ WAD S DRDQVNTRHY STSMRVALDRGEFFL V YQPIIRLADNRIIGAE
ALLRWEHPTLGTLL PGRFIDRAENNGL MVPLTAFVLEQACRHVRSWRDHSTDPQPFVSVNVSASTICDPG
FLVLVEGVLGETGLPAHALQLELAEDARLSRDEKAVTRLQELSALGVGIAIDDFGIGFSSLAYLPRLPVD
VVKLGGKFTECLDGDTQARLANEQTTRAMIDLODKLGTTVTAKLVESPSQA A RLR AFGCK A AQGWHF AK A
LPVDFFRE
[0208] SEQ ID NO: 18 [0209] 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.
1 ttgatcgact acgaagagat gtttaggggc gcgatgcaag cgcgagcgat ggtagccaat 61 cctgaccaat gggcggactc cgaccgcgac caggtcaaca ctcgccatta tctgtccact 121 tcgatgcgcg tggcactgga tcgcggtgaa ttcttcctcg tctaccagcc aatcatccgg 181 cttgccgaca accgcatcat cggcgccgag gccctgctgc gctgggaaca cccgacgttg 241 ggcacgclac teccgggccg gticatcgac cgtgecgaga acaaeggact gatgglgccg 301 ctcacggcct tcgtgctcga gcaggcctgc cgccacgtcc gcagttggcg tgaccacagc 361 accgacccgc aaccgtttgt cagcgtcaac gtctccgcca gcaccatctg cgatcccggc 421 ttcctggtgc tggtcgaagg tgtgctcggc gaaaccggcc tgcccgccca tgccctgcag 481 ctcgaactgg ccgaggacgc gcgccttagc agagacgaga aggcggtgac caggctacaa 541 gaattgtccg ctctcggcgt cggcatcgcc atcgacgact tcggcattgg attctccagc 601 cicgcctacc accecgcct ccccgtcgac gtggtcaaac tcgggggaaa gttcatcgag 661 tgcctcgatg gcgacattca agctcggctg gccaacgaac agatcacccg ggcaatgatc 721 gaccttggcg acaagctcgg tatcaccgtc actgcaaagc tagtcgaaag ccccagccaa 781 gccgcccggt tgcgcgcctt eggctgtaaa gccgcacaag gctggcactt tgccaaggca 841 ctgccggtcg acttatcag agagtag [0210] SEQ ID NO:19 [0211] Diguanylatc 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 Al 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 arc 100%
identical to the BCG diguanylate phosphodiesterase AHM07112.1.
MDRCCORATAFACALRPTKLIDY EEMFRGAMQARAMVAN PDQ WADSD RDQV N TRHY L ST S
MRVALDRGEFFLVYQPIIRLADNRIIGAEALLRWEHPTLGTLLPGRF IDRAENNGLMVPL
TAFVLEQACRHVRSWRDHSTDPQPITVSVNVSASTICDPGELVLVEG VLGETGLPAHALQL
ELAEDARL S RDEKAVTRLQEL SALGVGIAIDDF GIGF S SLAYLPRLPVDVVKLGGKFIEC
LDGDTQARL ANEQTTR A MIDLG DKLGITVTAKLVETPSQA ARLR AFGCK A A QGWHFA K A L
PVDF FRE
[0212] The sequences referenced in the application are summarized in Table 1 below.
Table 1 SEQUENCE DESCRIPTION
NUMBER
SR() ID NO:] 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-Phspoo::disA which overexpresses the disA gene, DNA
sequence SEQ ID NO:4 Bifunctional diguanylate cyclase/phosphodiesterase BCG R507340 from BCG and other related mycobacteria, amino acid sequence SEQ ID NO:5 Bifunctional diguanylate cyclase/phosphodiesterase BCG R507340 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 (IMP-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 (IMP-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- Ph.sp60::disA::C0cGAS: :rnCherry which overexpresses the disA gene, the codon-optimized human cGAS gene, and mChen-y, 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 TD NO:16 Bifuncticmal c-di-AMP & cGAMP pliosphodiesterase CdnP from M.
tuberculosis with 20 amino acid N-terminal extension, amino acid sequence.
SEQ ID NO:17 Diguanylate phosphodiesterase AII1V107112.1 from BCG and other related mycobacteria, amino acid sequence SEQ ID NO:18 Diguanylate phosphodiesterase AIIM07112.1 from BCCi and other related mycobacteria, DNA sequence SEQ ID NO:19 Diguanylate phosphodiesterase Ry1357c or MT1400 from Mycobacterium tuberculosis and BCG Pasteur 1173 P2 with 19 amino acid N-tenninal extension, amino acid sequence SEQ ID NO: 20 DNA sequence for the 1350 bp panCD operon from BCG Pasteur SEQ ID NO: 21 Protein sequence for the 301 aa PanC polypeptide from BCG Pasteur SEQ ID NO: 22 Protein sequence for the 139 aa PanD polypeptide from BCG
Pasteur SEQ ID NO: 23 DNA sequence for the 2501 bp panCD-containing region from BCG
SEQ ID NO: 24 Protein sequence for the 724 aa mutant PanC polypeptide from BCG

SEQ ID NO: 25 Protein sequence for the 139 aa PanD polypeptide from BCG Tice SEQ ID NO: 26 DNA sequence alignment of the BCG Pasteur and BCG Tice panC
SEQ ID NO: 27 DNA sequence alignment of the BCG Pasteur and BCG Tice panD
SEQ ID NO: 28 L primer to amplify the panCD operon (diagnostically) SEQ ID NO: 29 R primer to amplify the panCD operon (diagnostically) SEQ ID NO: 30 pSD5.hsp65-disA.Kan SEQ ID NO: 31 pSD5.hsp65-disA.panCDa.'Ã-No Kan SEQ ID NO: 32 DNA sequence for pJV53 (recombineering plasmid) SEQ ID NO: 33 DNA fragment containing panCD allelic exchange substrate cassette SEQ ID NO: 34 dif-Hyg-dif cassette SEQ ID NO: 35 pUC-Hyg Plasmid SEQ ID NO: 36 pUC-Hyg-panCD KO plasmid SEQ ID NO: 37 Left primer used to generate the backbone of "pSD5.hsp65-SEQ ID NO: 38 Right primer used to generate the backbone of "pSD5.hsp65-SEQ ID NO: 39 Left primer used to generate the panCD portion of "pSD5.1isp65-SEQ ID NO: 40 Right primer used to generate the panCD portion of "pSD5.hsp65-102131 In one embodiment, the present invention relates to an expression cassette or expression vector including 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 aspects, the expression vector or expression cassette further includes a nucleic acid sequence encoding a DNA
integrity scanning (disA) protein which functions as a diadenylate cyclase, or a functional part thereof. In other aspects, the nucleic acid sequence encoding a Rv1354c protein does not contain a phosphodiesterase gene or phosphodiesterase domain. In some aspects, the expression vector or expression cassette does not contain a phosphodiesterase gene or phosphodiesterase domain.
[0214] 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 includes 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 aspects, an expression vector or expression cassette of the invention includes one or more sequences operably linked to a nucleic acid of the invention which direct termination of transcription, post-transcriptional cleavage, and/or polyadenylation. In some aspects, an expression vector or expression cassette of the invention includes a variable length intervening sequence and/or a selectable marker gene operably linked to a nucleic acid of the invention.
[0215] In one embodiment, the present invention relates to a strain of Mycobacterium including an expression vector or expression cassette of the invention described herein. In some aspects, the strain of Mycobacterium is Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof. In other aspects, the strain of Mycobacteriwn is BCG. In some aspects, the strain includes the plasmid of SEQ ID NO:13.
102161 In another embodiment, the present invention relates to a strain of Mycobacteriwn 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 aspects, the expression or overexpression results in the 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 aspects, 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). In some aspects, the strain of Mycobacterium is Illycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof. In some aspects, the strain of Mycobacterium is BCG.
[0217] Statistically significant anti-tumor effects with BCG-di.sA-OE in the rat MNU bladder cancer model [0218] 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 Figure 7. The inventors have now performed the 16-week rat MNU model twice. Figure 7 was based on Experiment 1 and shows that BCG-disA-OE
displays a trend towards a better outcome versus BCG-WT. After performing Experiment 2 and combining its data with Experminent 1, it is now shown 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 Figure 15).
[0219] Reduction of tumor-suppressive Treg cells by BCG-disA-OE
in a murine syngeneic bladder cancer tumor model.
[0220] 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 a murine syngeneic bladder cancer tumor model using BBN975 cells was developped. 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 Figure 16, 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.
102211 BCG-disA-OE delivers sustained STING agonist from the intracellular compartment.
[0222] Persistence of BCG in the bladder.
[0223] 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.
[0224] Durek et al. (The fate of bacillus Calmette-Guerin after intravesical instillation. J Urol.
2001; 165: 1765-1768. PM1D 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 was 27.1% on day 7 immediately before the next instillation (FIG. 38). The investigators also evaluated bladder biopsies by PCR for mycobacterial DNA within 1 week after the 6th 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.
[0225] 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 microorganism 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.
[0226] BCG-disA -OE is safer than BCG-WT in two separate mouse models [0227] 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.
[0228] 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 Figures 17B, 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.

[0229] 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
[0230] Trained immunity. Trained immunity refers to thc 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).
[0231] 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.
[0232] 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.
[0233] 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 Figure 18, upon receiving the second stimulus, the immune response tested (secretion of IL-1 p) 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.
[0234] 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 showed 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.
[0235] BCG-Tice-disA-OE expresses much higher levels of the disA
gene than BCG-WT
[0236] As may be seen in Figure 21, 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-AACT 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.
102371 STING agonist oyerexpression BCG strains such as BCG-disA-OE elicit pro-inflammatory changes in signaling pathways and cytokine secretion profiles in multiple model systems.
[0238] 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. When the comparisons were not statistically significant, BCG-disA -OE gave the stronger of the two responses.

[0239] Figure 23 also shows that the elevation of type 1 IFN
secretion in both BCG-disA-OE
and BCG-WT is STING-dependent.
[0240] In summary, BCG-di.s,4-0E is a more potent stimulator of pro-inflammatory cytokine expression and proinflammatory pathway induction than BCG-WT
[0241] The table below summarizes the data:
[0242] Table 2:
Mouse BMDM in vitro IRF3 qRT-PCR BCG-disA-OE > BCG-WT Figure 22 Mouse BMDM, BMDC, J774 IFN-fi ELISA BCG-disA-OE > BCG-WT Figure 24 macrophage cell line in vitro Mouse BMDM; BMDC. J774 IL-6 ELISA BCG-disA-OE > BCG-WT Figure 25 macrophage cell line in vitro Mouse BMDM, BMDC, J774 TNF ELISA BCG-disA-OE > BCG-WT Figure 26 macrophage cell line in vitro Rat bladder cancer NBT-II line TNF, IFN-y ELISA BCG-disA-OE > BCG-WT Figure 27 in vitro Human bladder cancer RT4 line IFN-f3, IFN-y, ELISA BCG-disA-OE > BCG-WT Figure 28 in vitro TNF, IL-1f3 5637, RT4, NBT-II bladder TFN-fl qRT-PCR BCG-disA-OE > BCG-WT Figure 29 cancer cell lines In vitro Mouse lungs in vivo (different IFN-f3, IF N-7, IL- ELISA BCG-disA -OE > BCG-WT Figure 30 time points) 6, TNF
In vivo Mouse spleens in vivo IFN-f3; IFN-7 IL-ELISA BCG-disA-OE > BCG-WT Figure 31 (4 weeks). In vivo 6; TNF
[0243] A method to produce an antibiotic gene cassette-free recombinant BCG which overexpresses a STING agonist biosynthetic gene.
[0244] 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.
[0245] The mycobacterial genetic operon pan CD 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 B5 (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.
[0246] Genetic deletion ofpanCD in mycobacteria has been shown to yield mutant strains that can only grow in the presence of added pantothenate. As such they are auxotroph for pantothenate.
ApanCD mutants of Atvcobacteriuni 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.
[0247] The inventors disclose a detailed method for generating an unmarked (no antibiotic gene cassettes) ApanCD 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.
[0248] The inventors disclose a detailed method for generating a shuttle plasmid which harbors the mycobacterial pan CD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::di,sil construct which overexpresses the dinsA gene and releases excess STING agonist, c-di-AMP). The shuttle plasmid is capable of replication in E. colt 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.
[0249] In the final manifestation of this disclosure, the inventors show a method to introduce the antibiotic-cassette-free plasmid harboring the mycobacterial pan CD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA
construct) into an unmarked BCG ApanCD 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 forpanCD complementation from the plasmid.
[0250] 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 ApanCD 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.
[0251] BCG-Tice (ATCC 35743) is a natural pantothenate auxotroph.

[0252] The inventors disclose that the Mycobacterium hovis 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 change leads to a frameshift mutation after the 246th 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 475 amino acid nonsense polypeptide at its C-ten-ninus. 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 pan CD 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.
[0253] 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.

[0254] 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::diAA construct) directly into BCG-Tice (ATCC 35743).
[0255] 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.
[0256] In one embodiment, the present invention relates to a pharmaceutical composition including an expression vector, expression cassette, or strain of the invention described herein and a pharmaceutically acceptable carrier.
[0257] In another embodiment, 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 aspects, 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 aspects, individuals with a bladder cancer such as NMIBC are treated with a modulator of the type 1 interferon response, and in some aspects an individual with bladder cancer is provided a modulator of expression type 1 interferon expression, such as an inducer of its expression.
[0258] In certain aspects, 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.
[0259] 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 RV1 354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DneV) 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.
[0260] In particular aspects 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 intravesi cal chemotherapies such as m i to myc i n 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.
[0261] In some aspects, 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.
[0262] In one embodiment, the present invention relates to a method of treating and/or preventing cancer, including 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 aspects, the cancer is, for example, bladder cancer (e.g., non-muscle-invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor. In other aspects, the solid tumor is, for example, a sarcoma, carcinoma, or lymphoma.
[0263] 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.
[0264] Pharmaceutical Preparations [0265] Pharmaceutical compositions of the present invention include 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 (IMP-AMP synfhase (DncV) protein, or functional part thereof; a cyclic (IMP-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 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.
[0266] 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.
[0267] 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 aspects, the present invention (e.g., expression vectors, strains, or pharmaceutical compositions) can be administered intravenously, i ntraderm ally, tran sd erm ally, i ntrath ec ally, Intraarteri ally, i ntraperi ton eally, 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. TNFa 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).
[0268] 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.
[0269] 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 include 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.
[0270] 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.
[0271] In a specific aspect 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 the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0272] In further aspects, 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.
[0273] 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.
[0274] 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.
[0275] In certain aspects, pharmaceutical compositions may include, for example, at least about 0.1% of an active compound. In other aspects, the active compound may include 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 foimulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0276] 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.
[0277] A. Alimentary Compositions and Formulations [0278] 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.
[0279] In certain aspects, 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 JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P.
Santos CA, 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 MJ, Ni X, Waldman M, Ewig CS, Hagler AT. Derivation of class TI force fields. VI.
Carbohydrate compounds and anomeric effects. Biopolymers. 1998;45:435-68.
PMID: 9538697;
Hwang JS, Chac SY, Lee MK, Bac YH. Synthesis of sulfonylurca conjugated copolymer via PEO
spacer and its in vitro short-tell"' bioactivity in insulin secretion from islets of Langerhans.
Biomaterials. 1998;19:1189-95. PMID: 9720902; Hwang SJ, 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 phainfaceutically pure and substantially non-toxic in the amounts employed.
In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

[0280] 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.
[0281] 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 aspects, 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%.
[0282] B. Parenteral Compositions and Formulations [0283] 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, intrathccally, 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).
[0284] 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.
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. Patent 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.
[0285] 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 i ntraperi ton e al administration. In this conn ecti on, 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.
[0286] 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.
[0287] C. Miscellaneous Pharmaceutical Compositions and Formulations [0288] In some aspects 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. Phatmaceutical 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 include 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.
[0289] 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
[0290] 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 phaimaceutical 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.
[0291] Kits of the Disclosure [0292] 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.
[0293] 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 is more than one components 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 included 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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] Presented below are examples discussing enhancement of trained immunity by re-engineered BCG overexpressing the PAMP molecule cyclic di-AMP, contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
EXAMPLES

MATERIAL AND METHODS
[0301] Referring specifically to Figs 39-62, the following material and methods are provided to details the methods used to obtain the presented results.
103021 Bacterial strains and culture conditions:
[0303] Mycobacterium bovis (M. bovis) Bacillus Calmette- Guerin (BCG) Pasteur (BCG-WT
Pasteur) (a generous gift from Dr. Frank Collins [FDA] and identical to BCG-Pasteur provided by the Pasteur Institute to the Trudeau Institute in 1967 as TMC No. 1011) and commercially available BCG-Tice (Onco-Tice , Merck) were used for the generation of c-di-AMP
overexpressing recombinant BCG strains. Genomic DNA from Mycobacterium tuberculosis (M. tb) strain CDC1551 was used for PCR amplification of disA (MT3692/Rv3586). Single isolated bacterial colonies growing on 7H11 plates supplemented with oleic-albumin-dextrose-catalase (OADC) (Cat. B11886, Fisher Scientific) were picked and propagated in 7H9 Middlebrook liquid medium (Cat. B271310, Fisher Scientific) supplemented with (OADC) (Cat. B11886, Fisher Scientific), 0.5% glycerol (Cat. G55116, Sigma) and 0.05% Tween-80 (Cat. BP338, Fisher Scientific).
Cloning experiments were performed using E. coli strain DH5-a (Cat. 18258012, Fisher Scientific) and was routinely maintained in LB broth. For generation of disA
overexpressing BCG, an E. coil-mycobacterial shuttle vector (pSD5.hsp60) was used to clone M.tb gene MT3692 or Rv3586 under the strong mycobacterial promoter hsp60. Clones were confirmed by gene sequencing and were used for bacterial transformation by electroporation method. Recombinant strains were confirmed using colony PCR against kanamycin cassette, subjected to whole genome sequencing and qPCR
analyses. Details of all bacterial strains, plasmids and constructs are listed in Table 3.
[0304] Table 3:
Name Description/Source Bacterial strains M. tuberculosis strain Mtb-CDC1551 Wild-type M tuberculosis M. bovis BCG strains BCG Pasteur M. hovis BCG Pasteur BCG-disA-OE (Pasteur) BCG Pasteur strain overexpressing disA
(MT3692) of Mtb BCG Tice M. hovis BCG Tice BCG-disA-OE (Tice) BCG Tice strain overexpressing disA
(MT3692) of M.tb E. coil strain DH5-a Competent E. coli (High Efficiency) Cell lines Urinary bladder carcinoma cells RT4 (ATCC HTB-2Tm) Human low grade urothelial cancer 5637 (ATCC HTB-9Tm) Human high-grade urothelial cancer NBT-II (ATCC CRL-1655TM) N-butyl-N-(4-hydroxybutyl) nitrosamine induced tumor cell line in Rattus norvegicus Nara Bladder Tumor No. 2 MB49 (Cat. SSC148, EMD DMBA [7,12-dimethylbenz[a]anthracene]
induced murine Millipore) urothelial carcinoma cells, UPPL-1595 Luminal cell line established from a spontaneous primary bladder tumor in an Uroplakin-Cre driven PTEN/P53 knockout genetically engineered mouse model BBN 975 Basal- cell line established from, (,0 N-B tyl-N -(4-hydroxybu (813N) induced murine urothelial cancer model J28 (ATCC HTB-1Tm) high grade urothelial cancer Reporter cells RAW-Lucia TSG (invivoGen) TFN Reporter Raw 264.7 mu-rine macrophages Macrophage cell lines J774A.1 (ATCC TIB67Tm) Murine macrophage cell line Plasmids pSD5.hsp60 Mycobacterial expression plasmid with hsp60 promoter pSD5hsp60.MT3692 dis,4 over-expression plasmid Confocal Microscopy Reagents Primary Antibodies LC3B NB100-2220, Novus Biologicals P62/SQ SIMI P0067, Sigma Secondary Antibodies Goat anti-Rabbit IgG Alexa Fluor A32733, Thermo Fisher Scientific Plus Chemicals/Probes Fluorescein 5(6)-isothiocyanate 46950, Sigma (FITC) Hoechst 33342 62249, Thermo Fisher Scientific Flow Cytometry Reagents Antibodies (mouse BMDM study) anti-CD45 (clone 30-F11) Biolegend anti-CD124 (I clone 015F8) Biolegend anti-LA/1-E (clone 107630) Biolegend anti-Ly6C (clone HK1.4) Biolegend anti-CD1lb (clone M1/70) Biolegend anti-F4/80 (clone BM8) Biolegend anti-Ly6G (clone 1A8) Biolegend anti CD206 (clone C068C2) Biolegend anti-TNF (clone MP6-XT22) B iolegend anti- IL-10 (clone JES5-16E3) eBioscience Antibodies (HMDM study) anti-CD16 (clone 3G8) Biolegend anti-CD14 (clone 63D3) Biolegend an ti-HLA -DR (clone L243) Biolegend anti-CD! lb (clone ICRF44) Biolegend anti-TLR4 (clone HTA125) Biolegend anti-CD206 (clone 15-2) Biolegend anti-CD163 (clone (iHI/61) Biolegend anti-TNF (clone MAbl 1) Biolegend anti-IL-6 (clone MQ2-13A5) Biolegend Antibodies (myeloid cell panel, Syngeneic MB49 urothelial cancer model) CD45 (clone 30-F11) Biolegend CD124 (IL-4Ra) (clone I015F8) Biolegend I-a/I-e (clone M5/114.15.2) Biolegend F4/80 (clone BM8) Biolegend CD206 (clone C068C2) Biolegend) TNF (clone MP6-XT22) Thermo Fisher IL-10 (clone JES5-16E3) Thermo Fisher Antibodies (lymphoid cell panel, Syngeneic MB49 urothelial cancer model) CD45 (clone PerCP) Biolegend CD25 (clone PC61) Biolegend CD3 (clone 17A2) Biolegend CD4 (clone GK1.5) Biolegend CD8a (clone 53-6.7) Biolegend FOXP3 (clone MF-14) Biolegend Mouse IFN-y (clone XMG1.2) Biolegend FOXP3 (clone MF-14) Biolegend Reagents/Kits Protein transport inhibitor eBioscience, 00-4980-03 cocktail Zombie AquaTM Fixable Viability Biolegend, 423101 Kit TruStain FcXTM Biolegend, 101320 Fixation and Permeabilization Biolegend, 421403 Buffer Set Human TruStain FcXTM Biolegend, 422302 True-Stain Monocyte BlockerTm Biolegend, 426102 ELISA
Mouse ELISA Kits TNF- DuoSet DY410, R6000B, R and D Systems 1L-6 DuoSet DY406, R6000B, R and D Systems IFN- DuoSet DY485, R6000B, R and D Systems CCL2/JE/MCP-1 DuoSet DY479, R6000B, R and D Systems LEGEND MAXTM Mouse IFN-13 439407, Biolegend Human ELISA Kits TNF- DuoSet DY210, R6000B, R and D Systems 1L-6 DuoSet DY206, R6000B, R and D Systems IFN- ELISA Kit 41410-2, PBL Assay Science Rat ELISA Kits IFN- Quantikine RIFOO, R and D Systems TNF- Quantikine RTA00, R and D Systems 1L-2 Quantikine R2000, R and D Systems Chromatin Immunoprecipitation ChIP Antibodies Histone H3K9me3 (H3K9 (cat. A-4036-100, epigentek) Trimethyl) Polyclonal Antibody Anti-Histone H3 (tri methyl K4) (cat. ab8580, abeam) antibody - ChIP Grade ChIP Reagents BSA (Cat. A3294, Sigma-Aldrich) Salmon Sperm DNA (Cat. 15632011, ThermoFisher Scientific) IIEPES (Cat. 113375, Sigma-Aldrich) Formaldehyde (Cat. 252549, Sigma-Aldrich) EGTA (Cat. 03777, Sigma-Aldrich) EDTA (Cat. E6758, Sigma-Aldrich) TritonX-100 (Cat. T8787, Sigma-Aldrich) SDS (Cat. 71736, Sigma-Aldrich) NaHCO3 (Cat. 5761, Sigma-Aldrich) Nuclease free water (Cat. AM9930, ThermoFisher Scientific) SYBR green dye (Cat. 4385614, Applied Biosystems) [0305] Mammalian cell culture:
[0306] Cell lines: For cell-based in vitro infection assays J774.1 (American Type Culture Col I ection-ATCC T I B67 ' m, Manassas, VA, USA) murine macrophage cell lines were cultivated in RPMI-Glutamax (Cat. 61870-036, Fischer Scientific), supplemented with 10%
heat inactivated fetal bovine serum (FBS) (Cat. 10082147, Fischer Scientific) with 1%
streptomycin/penicillin at 37 C with 5% CO2. Urothelial carcinoma cell lines 5637 (ATCC HTB-9 rm), a human high grade urothelial cancer; RT4 (ATCC HTB-2Tm), a human transitional cell low grade urothelial cancer;
J82 (ATCC HTB-1Tm), a human high grade urothelial cancer; and NBT II (ATCC
CRL-1655Tm), N-butyl-N-(4-hydroxybutyl) nitrosamine induced tumor cell line in Rattus norvegicus Nara Bladder Tumor No. 2, UPPL1595 (luminal cell line established from a spontaneous primary bladder tumor in an Uroplakin-Cre driven PTEN/P53 knockout genetically engineered mouse model and were generously provided by Dr. William Kim (UNC Chapel Hill)., BBN975 (basal-cell line established from , 0.05% N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced murine urothelial cancer model and was generously provided by Dr. William Kim (UNC Chapel Hill), and MB49 (murine urothelial carcinoma cells, 7,12-dimethylbenz[a]anthracene (DMBA, EMD Millipore, Cat. SSC148) were maintained as monolayer in RPMTI 640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) with 1%
streptomycin/penicillin at 37 C with 5% CO2. Mouse fibroblast cell line NCTC
clone 929 [L cell, L-929, derivative of Strain L] (ATCCCD CCL-1TM) were routinely maintained as monolayer in DMEM media supplemented with 10% heat inactivated fetal bovine serum (FBS) with 1%
streptomycin/penicillin at 37 C with 5% CO2. All cell lines were not maintained more than 10 passage cycle and Mycoplasma testing was performed periodically while cells were in culture.
Reporter mouse cell line, RAW-Lucia ISG (InvivoGen, CA, USA) was cultivated in custom prepared media as per manufacturer's instructions.
[0307] Primary cells (Macrophages and Dendritic Cells):
[0308] For generation of murine bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs), bone marrow (BM) cells were isolated from 4-week old wild-type (WT) C57BL/6J (Charles River laboratories, North Wilmington, Mass) and STING-KO
mice (C57BL/6J-Tmem173gt/J, Jackson laboratories). Multiple vials of bone-marrow cells were preserved in cryopreservation media containing 10% DMS0 (Cat. D2650; Sigma) and 90% heat inactivated FBS (Cat. 10082147, Fischer Scientific) in liquid nitrogen. For differentiation of BM
cells into macrophages or DCs, random cryopreserved vials were chosen and differentiated for 6 days in BMDM-differentiation media made from DMEM containing 10% FBS, 1% MEM
amino acids (Cat. 11130051, Thermo Fisher Scientific), 1% MEM non-essential amino acids (Cat.
11140050, Thermo Fisher Scientific), 1% sodium pyruvate (Cat. 11360070, Thermo Fisher Scientific), 1% MEM vitamin (Cat. 11120052, Thermo Fisher Scientific) and antibiotics (Penicillin-Streptomycin solution) supplemented with 30% sterile mouse fibroblast L929 (ATCCO CCL-1TM) conditioned media. Differentiation of BM cells into DCs was carried out in low attachment 10 mm cell culture dish in presence of bone marrow-differentiation media in presence of recombinant murine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Cat. 315-03, Peprotech) for 48 h. Non-adherent cells were washed and loosely attached cells were allowed to differentiate into BMDCs for next 6 days. Cells were characterized for macrophage and DC markers using cell-surface staining and flow cytometry analyses. Human primary monocytes and human monocyte-derived macrophages (HMDMs) were used for cell-based in vitro infection assays. Peripheral blood-derived mononuclear cells (PBMCs) isolated from healthy male donors (leukopacks) aged between 18-30 were used for isolation of human monocytes (HM) or human monocyte-derived macrophages (HMDM). To separate blood constituents and isolation of buffy coat density gradient centrifugation (400 x g at 18 C for 30 min) of RPM1-1640 diluted blood over a Ficoll-PaqUeTM Plus reagent (Cat. 17-1440-02, GE Healthcare, Piscataway, NJ) was performed.
Cells were washed several times using 1 x PBS and were counted using hemocytometer. Once counted CD14+ human monocytes were isolated from PBMCs using magnetic labeling (Monocyte Isolation Kit II, Cat. 130-091-153, Miltenyi Biotec, San Diego, CA) and magnetic columns as per manufacturer's instructions. The purity of isolated CD14+ cells was confirmed using a fraction of cells stained with a fluorochrome-conjugated antibody against a monocyte marker as recommended by manufacturer and cells were analyzed using BD-LSR2 flow cytometer. Human monocytes were seeded (2.0 - 3.0 X 105 cells / ml in RPMI 1640 medium supplemented with 10%
FBS and 1% streptomycin/penicillin at 37 C with 5% CO2. Monolayers of CD14+
monocytes were differentiated into M1 [GM-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ) and IFN-y(20 ng/ml, PeproTech, Rocky Hill, NJ PeproTech)] or M2 [M-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ) and IL-4 (20 ng/ml, PeproTech, Rocky Hill, NJ PeproTech)] for next 7 days.
[0309] Animals:
[0310] Experimental procedures involving live animals were carried out in agreement with the protocols approved by the Institutional Animal Care and Use Committee (TACUC) at The Johns Hopkins University School of Medicine. For animal infection protocols, pathogen-free age 4-6 weeks female C57BL/6J (Charles River Laboratories, North Wilmington, Mass) and Fox Chase SCID mice (Charles River Laboratories North Wilmington, Mass.) were purchased and housed under pathogen-free conditions at an Animal Biosafety Level-3 animal facility without cross-ventilation. Fischer 344 female rats age 8 weeks (Harlan, avg. weight 160g) were housed at an BSL2 animal facility. Animals were given free access to water and standard chow and were monitored daily for general behavior and appearance by veterinary specialists.
[0311] In vitro infection assays:
[0312] For in vitro infection assays, cell lines or primary cells were seeded at required cell density in 6-well tissue culture plates or 10 mm petri dishes. For infection, log-phase wild-type and BCG-disA-OE strains were harvested by centrifugation and washed twice using DPBS to remove residual detergent and BSA then suspended in antibiotic-free RPMI 1640 media supplemented with 10% FBS. For infection assays, the bacteria were deposited at pre-calibrated multiplicity of infection (MOI). Infection was allowed for next 4 hours, followed by repeated washing of infected cells using warm DPBS to remove non-internalized bacteria.
Infected cells were incubated until endpoints in presence of RPMI-1640 medium supplemented with 10% FBS
and antibiotics.
[0313] Toxicity assays:
[0314] Human urothelial cancer cell lines, RT4, 5637, and J82, were cultured at 37 C under 5% CO2 in RPMI 1640 containing 10% FBS without antibiotics. For cell toxicity assay, 3000 cells for RT4 and 1500 cells for 5637 and J82 were seeded in a 96-well tissue-treated plate in triplicate, respectively. Twenty-four hours after seeding, cells were treated with the indicated ratio of BCG
to cells for 72 hours. To measure cell viability, CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) and FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany) were used according to manufacturer's protocols. Relative cell viability was calculated by dividing the viability of the indicated ratio by that of a control.
[0315] For Annexin-PI staining, 0.5 million J774.1 cell and BMDMs were plated per well in 6-well plates for physical attachment. Cells were exposed at 1:10 MOIs for 24 hours using wild-type and BCG-disA-OE strains of Tice and Pasteur to determine the BCG
cytotoxicity following exposure. At the endpoint of infection or treatment cells were non-enzymatically removed using 0.02% EDTA-PBS solution. Cells were washed twice with ice-cold PBS and FITC-annexin-PI
was done as per manufacturer's instruction using FITC Annexin V Apoptosis Detection Kit I (Cat.
556547, BD Biosciences). Flow cytometry was performed using a BD LSR II flow cytometer of the Flow Cytometry Core Facility at The Bloomberg School of Public Health, Johns Hopkins University). Data was processed using FlowJo software (Tree Star v10).
[0316] Quantitative real-time ca'CR:
[0317] Gene expression profiling was carried out using total RNA
isolated from cell lines or primary cells. For RNA isolation from rat bladders, pieces of whole bladder samples were excised, snap frozen in liquid nitrogen immediately after harvesting and stored in RNAlater (Cat. AM7021, Ambion) at -80 C. Total RNA isolation was carried out using RNeasy system (Cat. 74106, Qiagen). Real-time qPCR was performed using the StepOnePlus system (Applied Biosystems).

For gene expression analyses in cell lines and primary cells, SYBR Fast green double stranded DNA binding dye (Cat. 40g561 2, Applied Biosystems) was used. Gene expression analyses in rat bladder tissues were performed using TaqMan gene expression assays. Gene-specific qPCR
primers were purchased from Integrated DNA Technologies and all TaqMan gene expression assays were purchased from Thermo Fischer Scientific. Amplification of RNU6, 13-actin, GAPDH
were used as endogenous control for RNA samples derived from human, mouse and rat cells/tissues respectively. All experiments were performed at least in triplicate and data analyses was done using 2-AACT method. Details of NCBI gene identifiers and primer sequences are given in the Table 4.
[0318] Table 4:
Cloning primers used in the study Accession Gene Sequence (5'-3') HQ ID
NO:
Number pSD5hsp60.MT3692 GCiGCATCATATGCACGCTGTGACTCGTC SEQ
ID
(F) NO:107 pSD5hsp60.MT3692 GGGACGCGTTATTGATCGCTGATGGTCGATT SEQ ID
NO:42 (R) Kanamyein cassette (F) GAGAAAACTCACCGAGGCAG SEQ
ID NO:43 Kanamycin cassette (R) GTATTTCGTCTCGCTCAGGC SEQ
ID NO:44 32287254 M. tb sigH (F) GCGATGGTGGCTTCTCCCTCG SEQ
ID NO:45 M.tb sigH (R) CCATCTTGCACAGCTCGCGTAG SEQ
ID NO:46 qPCR primers used in the study Mouse Primers 11461 Mousc43 actin (F) TAAGGCCAACCGTGAAAAGATG SEQ
ID NO:47 Mouse.13 actin (R) CTGGATGGCTACGTACATGGCT SEQ
ID NO:48 21926 Mouse.TNF-a (F) GACCCTCACACTCAGATCATC SEQ
ID NO:49 Mouse.TNF-a (R) GCTGCTCCTCCACTTGGT SEQ
ID NO:50 15977 Mouse IFN-13 (F) CCACAGCCCTCTCCATCAAC SEQ
ID NO:51 Mouse.IFN-13 (R) CTCCGTCATCTCCATAGGGA SEQ
ID NO:52 16193 Mouse.IL6 (F) CTGCAAGAGACTTCCATCCAG SEQ
ID NO:53 Mouse.IL6 (R) CAGGICTGTIGGGAGTGG SEQ
ID NO:54 15978 Mouse.IFN (F) AGCGGCTGACTGAACTCAGATTGT SEQ
ID NO:55 Mousc.IFN (R) GTCACAGTTTTCAGCTGTATAGGG SEQ
ID NO:56 16176 Mouse.IL1 (F) GGAGAGTGTGGATCCCAA SEQ
ID NO:57 Mouse.IL1 (R) GTGGAGTTTGAGTCTGCAG SEQ
ID NO:58 20296 Mouse.MCP1 (F) GGCTCAGCCAGATGCAGTTAAC SEQ
ID NO:59 Mouse.MCP1 (R) GATCCTCTTGTAGCTCTCCAGC SEQ
ID NO:60 16160 Mouse.IL12b (F) GAAAGACGTTTATGTTGTAGAGG SEQ
ID NO:61 Mouse.IL12b (R) GACTCCATGTCTCTGGTCTG SEQ
ID NO:62 17329 Mouse.CXCL9 (F) GGAGTTCGAGGAACCCTAGTG SEQ
ID NO:63 Mouse.CXCL9 (R) GGGATTTGTAGTGGATCGTGC SEQ
ID NO:64 15945 Mouse.CXCL10 (F) GTGGGACTCAAGGGATCCCTCTC SEQ
ID NO:65 Mousc.CXCL10 (R) GCTTCCCTATGGCCCTCATTC SEQ
ID NO:66 18126 Mouse.NOS2 (F) GTTCTCAGCCCAACAATACAAG SEQ
ID NO:67 Mouse.NOS2 (R) GGAACATTCTGTGCTGTCCC SEQ
ID NO:68 20299 Mouse.CCL22 (F) CTCTGATGCAGGTCCCTATGGTG SEQ
ID NO:69 Mouse.CCL22 (R) GGCAGAGGGTGACGGATGTAG SEQ
ID NO:70 Human Primers 26827 Human. RNU6A (F) CTCGCTTCGGCAGCACATATAC SEQ
ID NO:71 Human. RNU6A (R) AATATGGAACGCTTCACGAATTTG SEQ
ID NO:72 3456 Human.IFN13 (F) CAACTTGCTTGGATTCCTACAAAG SEQ
ID NO:73 Human.IFN13 (R) TATTCAAGCCTCCCATTCAATTG SEQ
ID NO:74 3569 Human.IL6 (F) GGTACATCCTCGACGGCATCT SEQ
ID NO:75 Human.IL6 (R) GTGCCTCTTTGCTGCTTTCAC SEQ
ID NO:76 Rat Primers 64367 Rat.PPIB (F) CAGGATTCATGTGCCAGGGT SEQ
ID NO:77 Rat.PPIB (R) CCAAAGACCACATGCTTGCC SEQ
ID NO:78 24481 Rat.IFN-(3 (F) GAGTCTTCACACTCCTGGC SEQ
ID NO:79 Rat.IFN-(3 (R) GTCCTTCAGGCATGAGACAG SEQ
ID NO:80 298210 Rat.IFNLJ a (F) GCGTTCCTGCTGTGCTTCTC SEQ
ID NO:81 Rat.IFN-a (R) CCATTCAGCTGCCTCAGGAGC SEQ
ID NO:82 25712 Rat.IFNLJ y (F) CGTCTTGGTTTTGCAGCTCT SEQ
ID NO:83 Rat.IFN-y (R) CGTCCTTTTGCCAGTTCCTC SEQ
ID NO:84 24599 Rat. iNOS (F) 6 GTCiA GGGGA CTG(3 A CTTTTA G SEQ
ID NO:85 Rat. iNOS (R) TTGTTGGGCTGGGAATAGCA SEQ
ID NO:86 245920 Rat.IP10 (F) TCCACCTCCCTTTACCCAGT SEQ
ID NO:87 Rat.IP10 (R) AGAGCTAGGAGAGCCGTCAT SEQ
ID NO:88 24770 Rat.MCP-1 (F) CAGGICTCTGICACGCTTCTG SEQ
ID NO:89 Rat.MCP-1 (R) GCCAGTGAATGAGTACiCAGCAG SEQ
ID NO:90 25542 Rat.MIP-1 a (F) ACAAGCGCACCCTCTGTTAC SEQ
ID NO:91 Rat.MIP-1 a (R) CiCiTCAGGAAAATCiACACCCG SEQ
ID NO:92 24494 Rat.IL-1f3 (F) GACTTCACCATGGAACCCGT SEQ
ID NO:93 Rat.IL-1f3 (R) GGAGACTGCCCATTCTCGAC SEQ
ID NO:94 24835 Rat.TNF-a (F) CGTCCCTCTCATACACTGG SEQ
ID NO:95 Rat.TNF-a (R) CATGCTTTCCGTGCTCATG SEQ
ID NO:96 59086 Rat.TGF-fl (F) TGACGTCACTGGAGTTGTCC SEQ
ID NO:97 Rat.TGF-(3 (R) CCTCGACGTTTGGGACTGAT SEQ
ID NO:98 25325 Rat.IL-10 (F) CCTCTGGATACAGCTGCGAC SEQ
ID NO:99 Rat.IL-10 (R) TGCCGGGTGGTTCAATTTTTC SEQ
ID
NO:100 ChIP-PCR Primers Human.GAPDH (F) TACTAG CCi GITTT AC Ci G Ci CG SEQ
ID
NO:101 Human.GAPDH (R) TCCiAACAGGAGGA GCAGAGAGC GA SEQ
ID
NO:102 Human.IL-6 (F) CGGTGAA G AATGGA TGACCT SEQ
ID
NO:103 Human.IL-6 (R) A_AACC1AGACCCTTGCACAAC SEQ
ID
NO:104 Human.TNF-cx (F) ATC A GICAGTGGCCCAG,A,A G A CC C SEQ
ID
NO:105 Human.TNF-ct (R) CCAC7GICC7CGGATCATGCTTC_AG SEQ
ID
NO:106 [0319] ELISA:
[0320] Sandwiched ELISA was performed for cytokine (TEN-y, TNF-a, IL-6, IFN-13, IL-1[3 and MCP-1/CCL2) measurement in culture supernatants and animal tissues from lung, spleen or urinary bladder. Tissues and culture supernatants were flash frozen in liquid nitrogen immediately after harvest and stored at -80 C. Animal tissues were homogenized using micro tissue homogenizers (Cat. 1215D61, Kimble) and filter sterilized for measurement of various cytokine protein expression levels using sandwiched ELISA as per manufacturer's recommendations.
Details of all ELISA kits and accessory reagents are given in Table 4.
[0321] Multicolor confocal microscopy:
[0322] Multicolor laser confocal microscopy experiments were performed to determine phagocytosis, autophagy, and colocalization studies in urothelial cancer cells and primary macrophages. Cells were allowed to adhere on sterile glass cover slips placed in 6-well tissue culture plates and infections were carried at pre-calibrated MOT. Log phase bacterial cultures were labeled using FITC (Cat. F7250, Sigma). Following infection and treatment conditions, cells were fixed, permeabilized and blocked followed by overnight incubation with a primary antibody for LC3B (Cat. NB100-2220, Novus) or p62/SQSTM1 (Cat. P0067, Sigma-Aldrich) at recommended dilutions at 4 C. Cells were washed and incubated in the dark with Alexa Flour 647 conjugated secondary antibody (Cat. A32733, Thermo Fisher Scientific) at 4 C for lhour.
DNA staining was carried out using Hoechst 33342 (Cat. 62249, Thermo Fisher Scientific) for 5 minutes. Images were acquired using Zeiss LSM700 single-point, laser scanning confocal microscope at 63X
magnification at the Microscope Facility, Johns Hopkins School of Medicine.
Image processing and analyses was carried out using open source Fiji software. For LC3B or p62 quantification, perinuclear LC3B puncta (spot) was counted in a minimum 100 cells across different fields using and Tmaris 9.5Ø Quantification carried out using GraphPad Prism software.
[0323] Phaaocytosis assay:
[0324] IgG-FITC conjugated latex bead phagocytosis assay kit (Item No. 500290, Cayman Chemicals, USA) was used for phagocytosis studies. HMDMs were placed on sterile glass cover slip for attachment. Infection was carried out at 5:1 (HMDM versus BCG) ratio for 3 hours followed by addition of IgG-FITC beads in warm RPMI 1640 media at 1: 400 dilutions for 3 hours.
Nuclear staining was carried out using Hoechst 33342 (Cat. 62249, Thermo Scientific) and cells were visualized for bead phagocytosis using Zeiss LSM700 single-point, laser scanning confocal microscope. Quantification of beads was measured by mean fluorescence intensity (M.F.I.) calculations using open source Fiji Software.
[0325] Multicolor flow cNtometry:
[0326] The cell surface and intracellular staining was carried out on J774.1, murine BMDMs, human HMDMs and single cells derived from murine MB49 tumors and spleens. Flow cytometry panel were designed and if needed modified form murine myeloid and lymphoid cells and human myeloid cells. Details of all antibodies and the dilutions used are given in the Table 3. For in vitro infection assays, protein transport inhibitor cocktail (Cat. 00-4980-03, eBioscience) at recommended dilution, 12 hours before harvesting monolayer of cells. At the endpoint cells were harvested using a cell-detachment buffer (ice-cold PBS - 10 mM EDTA solution).
Single cell isolation was performed using animal tissues by harvesting tumors and spleens following necropsy. Briefly, tissues were manually disrupted before incubating in collagenase type I (Gibco) and DNase (Roche) in RMPI for 30 minutes at 37 C. Tumor and spleen cells were dissociated through a 70- m filter and washed with PBS. RBC lysis was performed for 5 minutes using ACK
lysis buffer (Cat. A1049201, Thermo Fisher Scientific) at room temperature.
Cells were washed twice using ice-cold PBS and stained using Zombie AqUaTM Fixable Viability Kit (Cat. 423101, Biolegend). Cells were washed and resuspended in FACS buffer (1% BSA, 2mM EDTA
in PBS), Fe blocked (TruStain FcXTM, Cat. 101320, and True-Stain Monocyte BlockerTM
Cat. 426102 Biolegend) and stained with conjugated primary antibodies as per manufacturer's protocol.
Intracellular staining was performed following fixation and permeabilization (Fixation and Permeabilization Buffer Set, eBioscience). Cells were washed and resuspended in flow buffer and acquired using BD LSRII with FACSDiva Software. analyses were performed using FlowJo (v10) (TreeStar).
[0327] The following antibodies were used to stain myeloid and lymphoid cells:
[0328] Mouse BMDMs: Anti-CD45 (clone 30-F11), anti-CD124 (clone 1015F8), anti-I-A/1-E
(clone 107630), anti-Ly6C (clone HK1.4), anti-CD1lb (clone M1/70), anti-F4/80 (clone BM8), anti-Ly6G (clone 1A8), anti CD206 (clone C068C2), anti-TNF (clone MP6-XT22) all Biolegend and anti- IL-10 (clone JES5-16E3 eBiosciences).
[0329] Human HMDMs: anti CD16 (clone 3G8), anti-CD14 (clone 63D3), anti-HLA-DR
(clone L243), anti-CD 1 lb (clone ICRF44), anti-CD206 (clone 15-2), anti-CD163 (clone GHI/61), anti-TNF (clone MAb11), and anti-TNF (clone MAbll) all Biolegend.
[0330] Mouse macrophages (syngeneic MB49 model of urothelial carcinoma): CD45 (clone 30-F11, Biolegend), CD124 (IL-4Ra) (clone 1015F8, Biolegend), I-a/I-e (clone M5/114.15.2, Biolegend), F4/80 (clone BM8, Biolegend), CD206 (clone C068C2, Biolegend), TNF
(clone MP6-XT22, Thermo Fisher), IL-10 (clone JES5-16E3, Thermo Fisher) [0331] Mouse T cells (syngeneic MB49 model of urothelial carcinoma): CD45 (clone PerCP, Biolegend), CD25 (clone PC61, Biolegend), CD3 (clone 17A2, Biolegend), CD4 (clone GK1.5, Biolegend), CD8a (clone 53-6.7, Biolegend), FOXP3 (clone MF-14, Biolegend), Mouse IFN-y (clone XMG1.2, Biolegend) and FOXP3 (clone MF-14 Biolegend).
[0332] hi vitro monoeyte trained immunity experiment:
[0333] In vitro training of primary human monocytes was performed as described ear1ier47.
PBMCs were isolated from healthy donors (leukopaks). Following magnetic separation, CD14+
monocytes were seeded in 10 1111113 tissue culture dishes for 3 hours in warm RPMI 1640 media supplemented with 10% FBS at 37 C with 5% CO2. Non-adherent cells were removed by washing cells using warm PBS. Monolayer culture of human monocytes was infected with BCG-WT and BCG-disA-OE strains at 5:1 (monocyte versus BCG) MOIs for 4 hours in presence of RPMI 1640 supplemented with 10% FBS. Non-internalized bacilli were washed out using warm PBS and subsequently incubated for 24 hours. Cells were again washed using warm PBS
and fresh warm RPMI 1640 media was added. For the following 5 days, cells were allowed to rest with a PBS
wash and addition of fresh media every 2nd day. Cells were re-stimulated on day 6 with RPMI
1640 supplemented with 10% FBS (negative control, without training) or TLR1/2 agonist, Pam3Cys (Cat. tlrl-pms, InvivoGen). Following stimulation, for 24 h, culture supernatants were collected, filter sterilized and quickly snap-frozen (-80 C) for cytokine measurement Cells were harvested for chromatin immunoprecipitation (ChIP) experiments to measure epigenetic changes on gene promoters.
[0334] Chromatin immunoprecipitation (ChIP): Human monocytes were fixed with a final concentration of 1% formaldehyde for 10 minutes at room temperature. Cell fixation was stopped using 125 mM glycine (Cat no. 50046, Sigma-Aldrich, USA), followed by sonication to fragment cellular DNA to an average size between 300 to 600 bp using Qsonica Sonicator Q125 (Cat.
15338283, Their/to Fisher Scientific). Sonicated cell lysates were subjected to immunoprecipitation (IP) by overnight incubation with recommended concentration of primary antibodies [(Histone H3K9me3 (H3K9 Trimethyl) Polyclonal Antibody cat. A-4036-100, epigentek); Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580), abeam)] in presence of magnetic Dynabeads (Cat no. 10004D, Thermo Fisher Scientific, USA) at 4 C.
Non-bound material was removed by sequentially washing the Dynabeads with lysis buffer, chromatin IP
(ChIP) wash buffer and Tris-EDTA (TE buffer). DNA elution was done using ChIP
elution buffer.
Amplification of different segments of the regulatory regions of immunity genes was carried out using qPCR using specific primers. Reactions were normalized with input DNA
while beads served as negative control. Details of all primary antibodies and sequence of primers have been given in Table 4.
[0335] Targeted Metabolite analysis with LC-MS/MS:
[0336] Targeted metabolite analysis was performed with liquid-chromatography tandem mass spectrometry (LC-MS/MS) as described ear1ier48. Metabolites from cells or snap-frozen xenograft tumor tissue were extracted with 80% (v/v) methanol solution equilibrated at ¨80 C, and the metabolite-containing supernatants were dried under nitrogen gas. Dried samples were re-suspended in 50% (v/v) acetonitrile solution and 4m1 of each sample were injected and analyzed on a 5500 QTRAP triple quadrupole mass spectrometer (AB Sciex) coupled to a Prominence ultra-fast liquid chromatography (UFLC) system (Shimadzu). The instrument was operated in selected reaction monitoring (SRM) with positive and negative ion-switching mode as described. This targeted metabolomics method allows for analysis of over two hundred of metabolites from a single 25-min LC-MS acquisition with a 3-ms dwell time and these analyzed metabolites cover all major metabolic pathways. The optimized MS parameters were: ESI voltage was +5,000V in positive ion mode and ¨4,500V in negative ion mode; dwell time was 3ms per SRM
transition and the total cycle time was 1.57 seconds. Hydrophilic interaction chromatography (HILIC) separations were performed on a Shimadzu UFLC system using an amide column (Waters XBridge BEH Amide, 2.1 x 150 mm, 2.5jtm). The LC parameters were as follows: column temperature, 40 C; flow rate, 0.30 ml/min. Solvent A, Water with 0.1% formic acid; Solvent B, Acetonitrile with 0.1% formic acid; A non-linear gradient from 99% B to 45% B in 25 minutes with 5min of post-run time. Peak integration for each targeted metabolite in SR1VI transition was processed with MultiQuant software (v2.1, AB Sciex). The preprocessed data with integrated peak areas were exported from MultiQuant and re-imported into Metaboanalyst software for further data analysis including statistical and principle components analyses.
[0337] Histologic analyses and immunohistochemistry (IHC):
[0338] For histologic analyses, a portion of bladder was formal in fixed and paraffin embedded.
Sections of 5vt in thickness on glass slides were stained with hematoxylin-eosin for classification according to the World Health Organization/International Society of Urological Pathological consensus as described ear1ier27. Tumor staging was performed by 2 board certified genitourinary pathologists (A.S.B., A.M.). Specimens were classified based on the percentage of involvement of abnormal tissue (1 = 10% involvement, 2 = 20% involvement, and so forth). For IHC staining, high-temperature antigen retrieval (18-23 psi/126 C) was performed by immersing the slides in Trilogy (Cell Marque). Endogenous peroxidase activity was blocked for 5 min in using Dual Endogenous Enzyme Block (Cat. S2003, Dako). Primary Antibodies used included Ki67 (1:50, Cat. ab16667; Abeam), CD68 (1:250, Cat. MCA341R; Serotec), CD86 (1:100, Cat.
bs-1035R;
Bioss) and CD206 (1:10K, Cat. ab64693; Abeam). For Ki67, slides were stained with ImmPACT
DAB (Vector Labs) for 3 min and counterstained with haematoxylin (Richard-Allen). Dual staining for CD68/CD206 and CD68/CD86 was achieved by first staining for CD68 with Impact DAB (Vector Labs) followed by secondary antigen retrieval and incubation as above with either CD86 or CD206 and visualized with ImmPACT AEC (Vector Labs). For each section, Ki67 expression was scored as a percentage of positive cells in the urothelium.
Dual stains for CD68/CD86 and CD68/CD206 were scored based on positive clusters of cells for each marker (0=

no staining, 1 = rare isolated cells positive, 2 = clusters of up to 10 positive cells, 3= clusters of >
positive cells).
[0339] In vivo experiments:
[0340] Intravesical BCG treatment in carcinogen induced NMIBC rat model:
[0341] The induction of urothelial cancer in rats and subsequent treatment of intravesical BCG
were performed. N-methyl-N-nitrosourea (MNU) instillations were given every other week for a total of 4 instillations. Fischer 344 female rats age 7 weeks (Harlan, avg.
weight 160g) were anesthetized with 3% isoflurane. After complete anesthesia, a 20G
angiocatheter was placed into the rat's urethra. MNU (1.5mg/kg) (Spectrum) dissolved in 0.9.% sodium chloride was then instilled and the catheter removed, with continued sedation lasting for 60 minutes to prevent spontaneous micturition and allow absorption. Eighteen weeks after the first MNU instillation, intravesical treatment with PBS or 5 x 106 CFU of each BCG strain (0.3m1 via a 20G angiocatheter) was administered weekly for a total of 6 doses. Rodents were sacrificed 2 d after the last intravesical treatment, and bladders were harvested within 48 hours of the last BCG instillation for mRNA and protein expression analysis as well as histological evaluation.
[0342] BCG infection of BALB/c mice and CFU enumeration:
[0343] To determine the lung bacillary burden of wild-type and BCG-disA-OE strains 6-week-old female BALB/c mice were exposed using the aerosol route in a Glasscol inhalation exposure system (Glasscol). The inoculum implanted in the lungs at day 1 (n=3 mice per group) in female BALB/c mice was determined by plating the whole lung homogenate on 7H11 selective plates containing carbenicillin (50 mg/ml), Trimethoprim (20 mg/ml), Polymyxin B (25 mg/ml) and Cycloheximide (10 mg/ml). Following infection, mice lungs were harvested (n =
5 animals/group), homogenized in their entirety in sterile PBS and plated on 7H11 selective plates at different dilutions. The 7H11 selective plates were incubated at 37 C and single colonies were enumerated at week 3 and 4. Single colonies were expressed at log CFU per organ.
[0344] SCID Mice time to death study:
[0345] The virulence testing of BCG-WT and BCG-disA-OE strains was done in severely compromised immunodeficient mice aerosol infection model as described previously. The inoculum implanted in the lungs at day 1 (n = 3 animals per group) was determined by plating the whole lung homogenate on 7H11 selective plates. For time to death analyses (n = 10 animals per group) infected animal were monitored until their death.
[0346] Syngeneic MB49 model of urothelial cancer:
[0347] MB49 tumor cells arc urothclial carcinoma line derived from an adult C57BL/6 mouse by exposure of primary bladder epithelial cell explant to 7,12-dimethylbenz[a]anthracene (DMBA) for 24 hours followed by a long-term cu1ture79. Before implantation, MB49 cells were cultured as monolayers in RPMI 1640 media supplemented with 10% FBS and 1%
streptomycin/penicillin at 37 C with 5% CO2. Cells were harvested using Trypsinization and cell viability was determined using Trypan blue dye. Live MB49 cells were resuspended in sterile PBS and adjusted at 1 x 105 live cells per 100 1. Female C57BL/6J mice, age 4-6 weeks (Charles River Laboratories) were subcutaneously injected with 1 x 105 MB49 cells in the right flank of hind leg. Tumor growth was monitored every 2nd day to observe the increase the tumor burden at the time of treatment initiation. Once palpable tumor developed (7 to 9 days, average volume ¨ 30 mm3), 1 x 106 bacilli of BCG-WT or BCG-disA-OE in a total 50 l PBS was injected intratumorally (Fig.
41). A total of 4 intratumoral injections of BCG was given every 3rd day. Tumors were measured by electronic caliper, and tumor volume was calculated using the following equation: tumor volume = length x width x height x 0.5326. Mice were killed at specified time, and tumors and spleens were collected after necropsy for single cell preparation.

BCG-DISA -OE ELICITS GREATER PRO-INFLAMMATORY
CYTOKINE RESPONSES IN MACROPHAGES THAN BCG-WT.
[0348] BCG-disA-OE is a genetically-engineered BCG strain in which an endogenous diadenylatc cyclase gene, disA, is fused to a strong promoter, leading to a 300-fold overcxpression of disA and a 15-fold increase in production of cyclic di-AMP (Fig. 39).
Compared with BCG-WT, BCG-disA-OE significantly increased STING pathway activation in macrophages as measured by IRF3 induction (Fig. 39). To control for the fact that numerous BCG strains are used worldwide and variabilities in their clinical efficacies have been described, two versions of BCG-disA-OE and a corresponding BCG-WT were generated: one using BCG-Tice and one using BCG-Pasteur. No significant differences between the Tice and Pasteur versions were detected.

[0349] To characterize the trained immunity-inducing potential of BCG-disA-OE versus BCG-WT, their capacity to induce cytokine expression in human monocyte-derived macrophages (HMDMs), primary murine bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC) as well as a macrophage cell line (J774.1) were evaluated. Consistent induction of IRF3, IFN-13, TNF-a and IL-6 in all myeloid cell types were found in response to BCG-disA-OE that was significantly higher than that seen with BCG-WT-exposed cells (Fig. 40 and Fig 43), and in human MDM and murine BMDM this difference was observed even in when cells were IFN-yprimed (Fig. 43). These differences were strictly STING-dependent as confirmed using BMDM from STING-/- mice (Fig. 40). Since STING activation leads to upregulation of NF-KB
via the TBK1-IRF3 pathway, it was found that expression of both TNF-a and IL-6 in the same panel of cells paralleled that of IFNI, and was significantly higher following exposure to BCG-disA-OE
compared with BCG-WT (Figs 41 and 43). Cyclic dinucleotides including cyclic di-AMP are known to be potent inducers of several chemokines (CXCL9, CXCL10 [TP-10], CXCL22, and MCP-1) as well as iNOS; consistent with this, IFN-y-primed BMDMs showed a more robust induction of these chemokines and iNOS when challenged with BCG-disA-OE strain than with BCG-WT (Fig. 41). The cellular toxicity was also assessed using annexin-PI
staining and found that whereas late apoptotic cell death remained at baseline with BCG-disA-OE
exposure in both BMDM and J774.1 macrophages, BCG-WT exposure elicited significantly higher levels of apoptotic cell death (Fig. 44) in the BMDM cells. These observations demonstrate that BCG-disA-OE elicits pro-inflammatory cytokine expression more potently than BCG-WT in primary human MDM as well as murine primary macrophages and macrophage cell lines.

PRO-INFLAMMATORY POLARIZATION OF MACROPHAGES
IS GREATER WITH BCG-D/SA-OE THAN WITH BCG-WT
[0350] Trained immunity is associated with polarization of macrophages towards inflammatory phenotypes with a concomitant shift away from anti-inflammatory states. To investigate macrophage polarization, flow cytometry was used to monitor phenotypic shifts of both murine and human primary macrophages following a 24 h exposure to BCG-disA-OE
or BCG-WT.
First, the MHC class II-expressing CD45 CD1 1 b F4/80 murine BMDM population were focused on, following in vitro BCG exposure using the gating scheme. As may be seen in Fig. 50 and Fig.

48, a significantly greater expansion of TNF-a-expressing CD1 1 F4/80-' (M1) murine BMDMs was observed following exposure to BCG-disA -OE than with BCG-WT. Next cells expressing the M2 surface receptors CD206f and CD124 among CD45f CD1 lb+ F4/80' macrophages were gated on and a greater reduction of this population with BCG-disA-OE than with to BCG-WT was observed (Fig. 50 and Fig. 49). Within this immunosuppressive cell population, there was a higher proportion of IL-10-expressing CD206+ CD124+ cells in BCG-WT-exposed macrophages, while IL-10-expressing cells were significantly reduced in response to BCG-disA-OE
exposure (Fig. 50 and Fig. 49). These results demonstrate that compared with BCG-WT, BCG-disA-OE
exposure elicits more extensive macrophage reprogramming with expansion of pro-inflammatory M1 macrophages displaying increased antigen presentation (MHC class II
expression) and TNF-a expression and contraction of immunosuppressive M2 macrophages expressing IL-10.
103511 Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells known to foster immunosuppression. Accordingly, the induction of monocytic-myeloid derived suppressor cells, M-MDSCs, was investigated (CD45-' Ly6Chi Ly6G-CD11b' F4/80) using primary murine BMDMs using the gating scheme shown.
Following BCG-WT exposure a significant expansion of M-MDSCs was observed, while in contrast this same population showed minimal expansion following BCG-disA-OE exposure (Fig. 50).
Moreover, the M-MDSCs elicited by BC G-WT exhibited higher IL-10 expression, whereas IL-10-expressing M-MDSCs were virtually absent after BCG-disA-OE exposure (Fig. 50). These observations suggest that BCG-WT contributes to an expansion of M-MDSCs which have immunosuppressive properties; however, forced overexpression of the pro-inflammatory PAMP cyclic di-AMP by BCG prevents M-MDSC expansion.
103521 The macrophage activation phenotypes in HMDMs isolated from several independent healthy human donors was next characterized. Both the BCG-WT and BCG-disA-OE
strains elicited increases in the population of classical macrophages (CD1lb CD14-' CD16), but these inductions were comparatively higher in response to BCG-disA-OE (Fig. 51 and Fig. 52).
classically activated antigen-presenting macrophages (CD14+ CD16- HLA-DR') and their ability to produce TNF-a or IL-6 were examined and it was found a significantly increased proportion of TNF-a and IL6- producing HLA-DR cells following exposure to BCG-disA-OE
compared to BCG-WT (Fig. 52 and Fig. 53). The M2 surface markers, CD206+ and CD163-', were also investigated on transitional or intermediate macrophages (CD11b+ CD14+ CD16+) and it was found a consistently greater decrease in them following BCG-disA-OE exposure than with BCG-WT
(Fig. 51, Fig. 53). The fraction of these intermediate macrophages expressing M2 surface markers and 1L-10 was also significantly lower in response to exposure to BCG-disA-OE
than with BCG-WT (Fig. 53). In summary, using both mouse and human primary macrophage ex vivo models, it was found that, compared with BCG-WT, BCG-disA-OE promotes greater macrophage activation towards an M1 phenotype (inflammatory), and concomitantly reduces the emergence of cells with immunosuppressive abilities, including M-MDSCs.

MACROPHAGES EXPOSED TO BCG-D/SA-OE
ARE MORE PHAGOCYTIC THAN THOSE WITH BCG-WT
[0353] Cyclic clinucleotides have been reported to recruit inflammatory macrophages which display high phagocytic potential. Consistent with these observations it waswe confirmed that HMDMs transfected with cyclic di-AMP showed increased phagocytosis and exhibited elongated dendrites compared to mock-transfected populations. It was then evaluated the phagocytic properties of HMDMs following exposure to the different BCG strains and found significantly greater phagocytosis of IgG-opsonized FITC-latex beads by macrophages exposed to BCG-disA-OE compared to BCG-WT (Fig. 54). In keeping with the previously established role of STING pathway activation in augmenting autophagy, it was found that a majority of intracellular BCG-disA-OE bacilli were co-localized with LC3B in IFN-y-activated primary BMDMs, while autophagy induction in BCG-WT was significantly lower. It was also found a significantly greater co-localization of BCG-cli sA-OE bacilli with the autophagy adapter protein p62 compared to that observed with BCG-WT. These results reveal BCG-disA-OE increases the levels of phagocytosis and autophagic processing within macrophages to a greater degree than BCG-WT, a phenomenon associated with enhanced peptide antigen presentation to MHC class-II
molecules.

BCG-D/SA-OE REPROGRAMS MACROPHAGES EPIGENETICALLY
AND POTENTIATES TRAINED IMMUNITY TO A GREATER DEGREE THAN BCG-WT
[0354] In light of recent data showing BCG to be a potent inducer of trained immunity through epigenetic modifications of key pro-inflammatory genes, it was hypothesized that the addition of cyclic di-AMP overexpression to standard BCG might potentiate epigenetic modifications in primary human monocytes. Having already established that BCG-disA -OE is a more potent inducer of macrophage TNF-a and IL-6 secretion than BCG-WT, it was confirmed this in primary human monocytes from a group of 6 healthy human subjects. The ability of traditional BCG to elicit trained immunity has been correlated with changes in epigenetic marks that increase pro-inflammatory gene expression. Thus, it was asked if the enhanced induction of TNF-a and IL-6 expression elicited by BCG-disA-OE compared with BCG-WT is epigenetically mediated. To this end, it was evaluated the promoter regions of the TNF-a and IL-6 genes for durable, antigen-independent epigenetic changes using an assay in which human monocytes exposed to BCG strains for 24 h were rested for five days prior to challenge with a heterologous antigen, the TLR1/2 agonist Pam3CSK4 on day 6 (Fig. 56). Using chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assays the activating histone methylation mark H3K4me3 present in the TNF-a and IL-6 promoters was quantified. It was observed that exposure to BCG-disA-OE led to greater enrichment of this mark than BCG-WT even without the heterologous second stimulation (i.e., adding RPMI media alone at day 6). Upon re-stimulation with Pam3CSK4 at day 6, the abundance of the activating epigenetic mark was further increased by both BCG
strains, but BCG-disA-0E-pretreatment yielded notably more enrichment than BCG-WT (Fig. 56).
Similarly, it was investigated the chromatin repression mark H3K9me3 at the same two promoters and found that, while both BCG strains led to reduced levels of H3K9me3 (which were further accentuated by addition of Pam3CSK4), the degree of reduction mediated by BCG-disA-OE was consistently greater than that mediated by BCG-WT, both upon initial exposure and after rest and re-stimulation (Fig. 56). Simultaneous measurement of TNF-a and IL-6 in BCG-trained culture supernatant following non-specific stimulation by Pam3CSK4 revealed that BCG-disA-0E-trained macrophages produced significantly higher levels of these pro-inflammatory eytokines than did those trained with BCG-WT. These results indicate that an augmented BCG which overexpresses the PAMP molecule cyclic di-AMP leads to significantly more robust epigenetic changes classically associated with trained immunity.

BCG-DLSA -OE REPROGRAMS THE MACROPHAGE IMMUNO-METABOLIC
STATE TOWARDS PRO-INFLAMMATORY SIGNATURES
TO A GREATER DEGREE THAN BCG-WT
[0355] BCG-training has been reported to stimulate glycolysis as well as the tricarboxylic acid cycle through glutamine replenishment with accumulation of fumarate. To address whether the addition of cyclic di-AMP overexpression alters the BCG-mediated metabolomic shifts, LC-MS was used to characterize key metabolites in primary human and murine macrophages exposed to the two BCG strains. HMDMs or BMDMs showed increased catabolic signatures (elevated intracellular glucose and lactate) to a greater degree following a 24 h exposure to BCG-disA-OE than with BCG-WT. Also, the TCA cycle metabolites itaconate and fumarate were also more elevated with BCG-disA-OE than with BCG-WT. These observations suggest greater catabolism of carbon substrates for ATP generation consistent with a pro -in fl ammatory bi oenergeti c profile in macrophages infected with BCG-disA-OE than with BCG-WT.
[0356] Excess tryptophan catabolism to kynurenine by tryptophan dehydrogenase and indoleamine 2,3-dioxygenase (IDO) has been strongly associated with immunosuppression, and IDO inhibitors have shown potential as immune activators in a variety of infectious and oncologic diseases. Kynurenine levels were dramatically lower in macrophages following BCG-disA-OE
exposure than those seen with BCG-WT, and as would be expected tryptophan levels were elevated by BCG-disA-OE while BCG-WT led to tryptophan levels comparable to the baseline seen with heat-killed BCG controls. Citrulline levels were also higher while putrescine levels were lower with BCG-disA-OE than BCG-WT suggesting that nitric oxide synthase-mediated conversion of argininc to NO (pro-inflammatory) and citrullinc was more strongly induced by BCG-disA-0E. Finally, it was of interest that itaconate, an isocitrate lyase inhibitor made by macrophages that has been shown to have antibacterial activity, was more potently induced by BCG-disA-OE than BCG-WT. Thus, compared with BCG-WT, BCG-disA-OE elicited a greater pro-inflammatory metabolomic signature with reduced kynurenine accumulation and increases in glycolytic metabolites, NOS products, and itaconate production.

FUNCTIONAL EFFICACY TN VIVO: BCG-DLSA -OE DEMONSTRATES
SUPERIOR IMMUNOTHERAPEUTIC OUTCOMES
IN RELEVANT ANIMAL MODELS OF TRAINED IMMUNITY
[0357] In addition to being used as a TB vaccine, BCG has served as a first-line immunotherapy for the treatment of non-muscle invasive bladder cancer (NMIBC) since the mid-1970s. Recent studies have indicated that BCG exerts its antitumor effects via a trained immunity mechanism. Having demonstrated that augmenting BCG with excess cyclic di-AMP
release leads to improved trained immunity parameters across a battery of in vitro assays, it was sought to determine if these effects could be demonstrated in vivo.
[0358] First, BCG-disA-OE versus BCG-WT was tested in a carcinogen-induced model of NMIBC in which intravesical therapies can be introduced into the bladder as they are in humans with non-invasive urothelial cancer. The rat tV-methyl-Ar-nitrosourea (MNU) model of bladder cancer (BC) is schematized in Fig. 57 In this model urothelial dysplasia develops at week 14 after the first intravesical instillation of MNU and by week 24 rats display a different forms of urothelial cancer severity including carcinoma-in-situ (CIS), papillary Ta (superficial), or higher-grade Tl -T2 urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human bladder cancer. Following carcinogen-mediated tumor induction with 4 cycles of MNU (wk 0, wk 2, wk 4, wk6), groups of rats were treated with 6 weekly doses of intravesical BCG-disA-0E, BCG-WT, or no treatment from week 18-23. Upon sacrifice at wk 24 the rat urinary bladders were divided into halves for (i) RT-PCR analysis, and (ii) histologic analysis including tumor staging by a blinded genitourinary pathologist.
Transcriptional analysis of the whole excised bladders at week 24 showed that compared with BCG-WT, BCG-disA-OE elicited significantly increased levels of IFN-P, IFN-y, TNF-a, IL-113, CXCL10, MCP-I, MIP-la, and iNOS transcription while mRNA levels of the immunosuppressive cytokines IL-10 and TGF-p were reduced by both BCG strains (Fig. 57). These patterns of cytokine expression were confirmed at the protein level using ELISA for TNF-a 1L-2, and IFN-y and noted that intravesical BCG-disA-OE, strongly increased the levels of IFN-y in rat spleens while BCG-WT did not. Correspondingly, it was found a significant decrease in highest pathology grade, tumor involvement index and highest tumor stage (Fig. 57) in rats treated with BCG-disA-OE in comparison to untreated. By tumor involvement index BCG-disA-OE was statistically significantly superior to no treatment (p <0.001) and to BCG-WT (p < 0.05), whereas BCG-WT showed only a trend towards improvement over no treatment. Importantly, the highest tumor stage observed in BCG-disA-0E-treated rats was CIS, whereas it was Ti in those receiving BCG-WT, and T2 in untreated rats, and 53.3% of BCG-disA-0E-treated rats were cancer free (p=0.009) compared with 31.2% of BCG-WT and 0%
of the untreated rats (Fig. 57). Immunohistochemical analyses revealed a significant reduction in Ki67 staining in BCG-disA-0E-treated MNU rat bladders when compared to untreated (p < 0.01) and BCG-WT (p < 0.05) suggesting reduced tumor proliferation. CD68 staining of rat bladder showed significantly higher levels of macrophage recruitment with a trend toward elevation of the pro-inflammatory Ml-like CD86+ macrophages and a significant reduction in CD206+ M2-like macrophages that are associated with tumor promotion in the BC G-disA-0E-treated rats compared with untreated controls. These observations indicate that the enhanced induction of type I IFN and other proinflammatory signatures in bladders of tumor-bearing rats treated with BCG-disA-OE
correlated with the enhanced antitumor activity of the recombinant BCG strain.
[0359] The functional efficacy of BCG-disA-OE was also tested in a murine heterotopic, syngeneic bladder cancer model using MB49 urothelial cancer cells. Following flank engraftment with MB49 tumor cells, mice received four intratumoral treatments over 9 days as shown in Fig.
61. In this model BCG-disA-OE also showed more robust immunotherapeutic efficacy than BCG-WT as measured by tumor volume and weight after intraturnoral injection of BCG-disA-OE when compared with BCG-WT (Fig. 61). Histopathology demonstrated extensive necrosis and congestion in MB49 tumors treated with BCG-disA-OE when compared to BCG-WT and untreated. There were no significant changes in body weights of mice receiving BCG, however splenic weight was significantly increased by both BCG strains. We further characterized the impact of the treatments on macrophage polarization and recruitment of activated T cells in the tumor microenvironment (TME). As shown in Fig. 61, compared with BCG-WT, BCG-disA-OE
significantly reduced the abundance of immunosuppressive M2 macrophages when compared to untreated and BCG-WT and significantly (p < 0.01) increased proinflammatory M1 macrophages.
Similarly, BCG-disA-OE recruited significantly more IFN-y-producing CD4 T
cells when compared to BCG-WT, and both BCG strains increased IFN-y-producing CD8 T
cells. While both BCG strains recruited more CD4' and CD S' cells to the tumors, BCG-disA-OE uniquely recruited more CDS+ T cells to the spleens of treated animals. BCG-disA-OE
also significantly reduced tumor-associated T-regulatory (Treg) cells to a greater degree than BCG-WT in both tumor and spleen. In keeping with our earlier findings in primary cells, we also found that compared with BCG-WT, BCG-disA-OE elicited more potent cytokinc responses and autophagy in human urothelial cancer cells representing various tumor stages. These results indicate that in this murine model of urothelial cancer, BCG-disA-OE has superior antitumor efficacy than BCG-WT, and its efficacy correlates with shift in polarization of macrophages to Ml, increased activation of both CD4 and CD8' T cells, and a reduction of local intratumoral and systemic Treg cell populations.

SAFETY: BCG-D/SA-OE IS LESS PATHOGENIC THAN BCG-WT
IN TWO MOUSE MODELS
[0360]
To address concerns that the enhanced pro-inflammatory immune responses elicited by BCG-disA-OE might lead to adverse effects, safety in two separate mouse models was evaluated.
An immunocompetent BALB/c mouse model of aerosol exposure was used and measured the lung bacillary burden after four weeks when adaptive immune responses are maximal (Fig. 58). While the day 1 implantation of the two BCG strains was equivalent, we observed that BCG-disA-OE
proliferated in murine lungs to a significantly lower degree than BCG-WT by a margin of 0.43 logio colony forming units (Fig. 58). As previously observed in cell-based models, pm-inflammatory cytokine levels in both lungs and spleens were significantly higher in BCG-dis,4-OE-exposed mice than those receiving BCG-WT (Fig. 60).
the two strains in immunocompromised SCID mice which do not survive infection with BCG were also tested.
Again, using a low dose aerosol exposure model (Fig. 59), it was observed a statistically significant survival prolongation with BCG-disA-OE compared to BCG-WT (Fig. 59). Thus, despite eliciting more profound inflammatory signatures in numerous model systems, BCG-disA-OE
is less pathogenic than BCG-WT in these two murine model systems.

DISCUSSION
[0361]
Numerous recombinant BCG strains have been generated and tested over the years.
These studies were generally conducted with the goal of improving either TB
protective efficacy or bladder cancer immunotherapy, but in certain cases the goal has been prevention of other infectious diseases. A common strategy has been to overexpress an antigen to elicit disease-specific immunity or a cytokine gene to boost local host responses. While many modified BCGs have shown efficacy in pre-clinical models, few have progressed to human clinical trials. To date only BCGAureC::hly (VPM1002), a BCG designed to enhance phagosome permeability and exposure of BCG antigens to cytosolic MHC class I antigen processing, has advanced to late stage clinical trials for tuberculosis. This is the first to specifically re-engineer BCG with the specific goal of improving trained immunity by overexpressing the PAMP molecular cyclic-di-AMP to increase STING pathway engagement.
[0362] To determine if trained immunity parameters may be increased, BCG-disA-OE versus BCG-WT were tested in a battery of in vitro assays. Cytokine release profiles, macrophage polarization, autophagy, phagocytosis, epigenetic modifications, and metabolic remodeling in -human and murine primary cells were evaluated. In each assay system, BCG-disA
-OE was a more potent potentiator of pro-inflammatory responses than BCG-WT. the cyclic-di-AMP expressing BCG was further tested in a functional in vivo assay of trained immunity, namely bladder cancer immunotherapy. In two separate models of urothelial cancer, BCG-disil-OE has greater immunotherapeutic efficacy than did BCG-WT indicating that our in vitro results were predictive of functional efficacy in a relevant animal model. Interestingly, despite eliciting a significantly more potent pro-inflammatory responses in our in vitro assay systems, BCG-disA-OE did not produce excess pathogenicity in two animal models of BCG infection or BCGosis.
[0363] It was also observed that BCG-WT did not uniformly elicit pro-inflammatory responses. For example, it was observed that treatment of murine macrophages with BCG-WT in fact induced a higher percentage of M-MDSCs (anti-inflammatory) compared with untreated controls (Fig. 50), and similarly BCG-WT led to elevated levels of the anti-inflammatory metabolite kynurenine. These findings of certain anti-inflammatory consequences of BCG-WT
may correlate with the observation that in countries which routinely use BCG
for TB prevention, vaccinees display reduced levels of asthma and atopic dermatitis. In contrast, this expansion of M-MDSCs in macrophages by BCG-WT was reversed by cyclic di-AMP overexpression which is in keeping with recent studies showing that STING pathway activation reduces the induction of MDSCs in certain cancers.

[0364] Trained immunity changes elicited by BCG may underlie the immunotherapeutic effects of BCG in cancer prevention. Therefore, another goal of this study was to evaluate whether the salutary effects of BCG-di.sA-OE as a NMIBC immunotherapy is mediated through engagement of STING pathway and modulates BCG-mcdiated trained immunity. In a rat model of NMIBC, it was found that whereas invasive tumors developed in untreated tumor-bearing rats (highest tumor grade of T2) as well as BCG-WT-treated animals (highest tumor grade of Ti), invasive bladder cancer was completely absent in rats treated with BCG-disA-0E. Similarly, in the MB49 mouse model of bladder cancer, BCG-dis24-0E was superior to BCG-WT in reducing tumor growth with associated increase in tumor necrosis, and these effects were accompanied by significantly higher recruitment of M1 macrophages, IFN-y-producing CD4 cells, and reduced accumulation of Treg cells in the tumors. Elevated levels of pro-inflammatory cytokines and chemokines were observed in bladders from tumor-bearing animals treated with BCG-disA-OE
compared to BCG-WT. Since non-immune cells have also been shown to possess immunological memory, the possibility that this cytokine response may have originated from myeloid cells in the TME and/or the tumor cells themselves was considered. Indeed, it was found that compared with BCG-WT, BCG-di.sA-OE elicited more potent cytokine responses in both primary macrophages and human urothelial cancer cells representing various tumor stages. This appeared to be a downstream consequence of STING activation since we found dramatically reduced expression in BMDMs from STING' mice. In addition, robust induction of several chemokines as has been observed in other studies with stimulation using exogenous STING agonists was found.
[0365] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
[0366] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims (16)

WHAT IS CLAIMED IS:

1. A method of suppressing the expression of myeloid-derivcd suppressor cells (MDSCs), M2 macrophages, and Treg cells in a tumor and inducing the expression of macrophages, dendritie cells (DCs), and T effector cells in a tumor 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 a tumor;
suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor;
and inducing the expression of macrophages, dendritic cells (DCs), and T effector cells in the tumor, thereby suppressing the expression of MDSCs, M2 macrophages, and Treg cells and inducing the expression of macrophages, DCs, and T effector cells in the tumor.

2. The method of claim 1, wherein suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor is observed when compared to the expression of MDSCs, M2 macrophages, and Treg cells in a tumor of a referenced subject not administered a pharmaceutical cornpositi on comprising the strain of Mycobacteri a.

3. The method of claim 1, wherein inducing the expression of macrophages, dendritic cells (DCs), and T effector cells in a tumor is observed when compared to the expression of macrophages, dendritic cells (DCs), and T effector cells in a tumor of a referenced subject not administered a pharmaceutical composition comprising the strain of Mycobacteria.

4. The method 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"-3'cGAMP; 2'-3'cGAMP and a combination thereof.

5. The method of claim 1, wherein the vector comprises a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rvl 354c protein, or a functional part thereof a second nucleic acid sequence encoding a 3'-3'cyclic GMP-AMP
synthasc (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.

6. The method of claim 1, wherein the tumor is a cancer selected from the group consisting of epithelial cancers, breast cancer, non-muscle invasive bladder cancer, melanoma, and a combination thereof.

7. The method of claim 6, wherein the tumor is a 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.

8. The method of claim 6, wherein the tumor is non-muscle invasive bladder cancer and is a BCG-naive non-muscle invasive bladder cancer (BCG-naive NMIBC) and the pharmaceutical composition is administered by intravesical instillation.

9. The method of claim 6, wherein the tumor is an epithelial cancer is selected from the group consisting of colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngcal cancer, endobronchial cancer, and a combination thereof and the pharmaceutical composition is administered to a luminal surface of the epithelial cancer.

10. The method of claim 1, wherein the tumor is a solid tumor and the pharmaceutical composition is administered by intratumoral, intravenous, intradermal, transdermal, intravesical topical, intramuscular or subcutaneous injection.

11. The method of claim 1, further comprising the step of administering a checkpoint inhibitor.

12. The method of claim 11, wherein the checkpoint inhibitor is selected from the group consisting of ipilimumab (anti-CTLA-4 antibody), nivolumab (anti-PD-1 antibody), pembrolizumab (anti-PD-1 antibody), cemiplimab (anti-PD-1 antibody), atezolizumab (anti-PD-Ll antibody), avelumab (anti-PD-Ll antibody), durvalumab (anti-PD-Llantibody ) and a combination thereof.

13. The method of claim 1, wherein the induced macrophages are M1 macrophages.

14. The method of claim 1, wherein the T effector cells are CD4+T cells.

15. The method of claim 1, wherein the T effector cells are CD8+ T cells.

16. The method of claim 1, wherein the tumor is a liquid tumor and the pharmaceutical composition is administered by intravenous, intradermal, transdermal, intravesical topical, intramuscular or subcutaneous injection.