CN115443143A - Recombinant therapeutic intervention for cancer - Google Patents

Abstract

Methods of inhibiting 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 are described. A pharmaceutical composition comprising a mycobacterium strain comprising an expression vector of the present invention is administered to a subject.

Description

Recombinant therapeutic intervention for cancer

Cross Reference to Related Applications

This application claims benefit of priority from U.S. patent application No. 16/790,161 filed 2/13/2020, which is a partial continuation-in-app (CIP) filed 2/638,943/2020/13/which is 35u.s.c. § 371 to international application PCT/US2019/022341, into the US national phase, international application date 3/14/2019, which claims benefit from U.S. provisional application No. 62/658,661 filed 4/17/2018, each of which is incorporated herein by reference in its entirety.

Incorporation of sequence listing

The materials in the accompanying sequence listing are hereby incorporated by reference into this application. The accompanying sequence Listing text file name is JHU4280_2WO _ sequence _ _Listing.txt, created at 11/2/2021, and is 157kb. The file may be evaluated using Microsoft Word on a computer using the Windows OS.

Statement of government interest

The invention was made with U.S. government support in accordance with approval numbers AI036973, AI037856 awarded by the National Institutes of Health. The united states government has certain rights in the invention.

Background

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 treatment, risk of progression to late stages, and the highest lifetime treatment of all cancers. Intravesical BCG (bacillus Calmette Guerin) instillation has been the standard of care treatment for NMIBC for 30 years. Intravesical BCG instillations are effective in 60% to 70% of patients. BCG has been shown to be a very effective vehicle for delivering antigens. Many studies demonstrating the propensity of the potential immune response to type I interferons and Th 1-induced mediated immune responses show promise. Efforts to generate recombinant BCG (rBCG) strains for NMIBC have focused on developing strains that enhance these anti-tumor immune responses. To date, such efforts have not resulted in significant improvements over traditional BCG.

Summary of The Invention

One embodiment of the invention provides a vector comprising a nucleic acid sequence expressing a protein or functional portion thereof that produces a STING agonist, including, by way of example, 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, a product of Vibrio cholerae (Vibrio cholerae) DncV protein); 2'-3' cGAMP (also known as 2'-5',3'-5' cGAMP, a product of human cGAS protein) and combinations thereof. Some vectors of the invention comprise a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA Integrity Scanning (DISA) protein or a functional portion thereof, and combinations thereof. Each of these nucleic acid sequences expresses a protein that produces one or more STING agonists as described in the definitions section of the specification. Some vectors of the invention include a fifth nucleic acid sequence encoding a PanC protein and a PanD protein, or functional portions thereof, in addition to one or more of the sequences listed above. Vectors comprising nucleic acid sequences encoding the PanC and PanD proteins or functional portions thereof typically do not contain an antibiotic resistance gene. Suitable vectors for use in the present invention may include vectors which replicate episomally in more than one copy, or vectors which integrate into the bacterial chromosome in a single copy or are otherwise present in the bacterial cell. The vectors of the invention may be stably integrated into the bacterial genome, or they may be stably replicated as episomal plasmids (episomal plasmids). Suitable third nucleic acid sequences include those that overexpress the cyclase domain of the cyclic GMP-AMP synthase (cGAS) protein. Other suitable third nucleic acid sequences may express a non-functional cyclic GMP-AMP synthase (cGAS) protein having the ability to recognize regulatory DNA. The vector of the present invention may further comprise a nucleic acid sequence encoding a sequence or protein that knocks out the expression of the PDE gene of the mycobacterium (mycobacterium) strain used in the present invention.

Another embodiment of the invention provides a mycobacterium strain comprising any one of the vectors of the invention, including a vector comprising a nucleic acid sequence that expresses a protein, or functional portion thereof, that produces a STING agonist. As mentioned above, examples of STING agonists include 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, a product of the Vibrio cholerae DncV protein); 2'-3' cGAMP (also known as 2'-5',3'-5' cGAMP, which is the product of human cGAS protein) and combinations thereof. Examples of suitable nucleic acid sequences include nucleic acid sequences selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA Integrity Scanning (DISA) protein or a functional portion thereof, and combinations thereof. Examples of suitable strains of Mycobacterium (Mycobacterium) for use in the present invention include, for example, mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium bovis (Mycobacterium bovis), or combinations thereof. Another strain used in the present invention is Mycobacterium Calmette-Guerin (BCG). The mycobacterium strain used in the present invention may be a pantothenate (pantonate) auxotroph of BCG lacking its panCD gene operon. The panCD auxotrophic strain lacks genomic sequences capable of encoding functional PanC and/or PanD proteins. In some embodiments, a mycobacterium strain that is pantothenate auxotrophic comprises a vector of the present invention comprising a panCD nucleic acid encoding a PanC and PanD protein, or functional portions thereof. The vectors of the invention comprising the panCD nucleic acid sequences preferably do not contain an antibiotic resistance gene or a nucleic acid sequence encoding a functional protein that provides antibiotic resistance. The mycobacterium which is pantothenate auxotrophic for the present invention preferably does not contain an antibiotic resistance genomic gene or does not encode a functional protein that provides antibiotic resistance.

Another embodiment of the invention provides a pharmaceutical composition comprising any one of the mycobacterium strains of the invention and a pharmaceutically acceptable carrier.

Another embodiment of the present invention provides a method of eliciting a type 1 interferon response, enhancing expression of pro-inflammatory cytokines, and/or eliciting a trained immunity (trained immunity) in a subject, the method comprising the steps of: administering a pharmaceutical composition comprising any one of the strains of the invention into a subject; and eliciting a type 1 interferon response, enhancing expression of a pro-inflammatory cytokine, and/or eliciting a training immunity in the subject. In one aspect, the pharmaceutical composition is administered into the bladder of the subject via a catheter.

Another embodiment provides a method of treating or preventing cancer in a subject using a mycobacterium strain of the present invention. The method comprises the following steps: administering to a subject having cancer a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a protein that produces a STING agonist, or a functional portion thereof; and treating or preventing cancer in a subject. By way of example, the invention may be used to treat or prevent cancer, including epithelial cancer, breast cancer, non-muscle invasive bladder cancer. In some aspects, the cancer is BCG-unresponsive non-muscle invasive bladder cancer (B)CG non-responsive NMIBC) and the pharmaceutical composition is administered by intravesical instillation. In some aspects, the cancer is BCG untreated non-muscle invasive bladder cancer ((ii))

non-muscle innovative radiator (BCG untreated NMIBC) and the pharmaceutical composition is administered by intravesical 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, bronchial cancer, and combinations thereof, and the pharmaceutical composition is administered to the luminal surface of the epithelial cancer. In some aspects, 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 invention may include the step of administering a checkpoint inhibitor, such as, by way of example, an anti-PD 1 antibody, an anti-PDL 1 antibody, or a combination thereof. In another aspect, the cancer is bladder cancer and the pharmaceutical composition is administered via a catheter.

One embodiment of the present invention provides an expression vector comprising: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (dis a) protein or functional portion thereof that functions as a diadenyl cyclase, or a combination thereof. Some expression vectors of the invention comprise a first nucleic acid sequence which overexpresses the cyclase domain of the Rv1354c protein when compared to the expression of the native Rv1354c protein as a reference. Some expression vectors of the invention comprise a second nucleic acid sequence that overexpresses a cyclic GMP-AMP synthase (DncV) protein when compared to the expression of a native DncV protein. Some expression vectors of the invention comprise a third nucleic acid sequence that overexpresses the cyclase domain of the cyclic GMP-AMP synthase (cGAS) protein when compared to expression of the native cGAS protein. Suitable Rv1354 proteins for use in the present invention include mycobacterium tuberculosis Rv1354 protein. Suitable DncV proteins for use in the present invention include the Vibrio cholerae DncV protein. Suitable cGAS proteins for use in the present invention include Homo sapiens (Homo sapiens) cGAS protein. Suitable DISA proteins for use in the present invention include Mycobacterium tuberculosis DISA proteins.

Another embodiment of the invention provides a strain of BCG comprising a cdnP gene, an Rv1354c gene, an Rv1357c gene or a combination thereof, wherein the cdnP gene is incapable of expressing a functional cyclic dinucleotide phosphodiesterase (cdnP) protein, the Rv1354c gene is incapable of expressing a functional Rv1345c protein, and/or the Rv1357c gene is incapable of expressing a functional Rv1357 protein. Some BCG strains of the invention may have an Rv1354c gene comprising a non-functional EAL domain. The BCG strain of the invention may comprise any expression vector of the invention.

Another embodiment of the present invention provides a method of treating or preventing bladder cancer, the method comprising the steps of: administering a pharmaceutical composition comprising a BCG strain comprising an expression vector of the invention into the bladder of a subject; and treating or preventing bladder 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 by catheter.

Another embodiment of the present invention provides a method of eliciting a type 1 interferon response in a subject, the method comprising the steps of: administering a pharmaceutical composition comprising a BCG strain comprising an expression vector of the invention into a subject, such as the bladder of a subject; and enhancing a type 1 interferon response in the subject compared to a reference subject not administered the pharmaceutical composition.

Another embodiment of the present invention provides a method of treating or preventing cancer in a subject, the method comprising the steps of: administering a pharmaceutical composition comprising a BCG strain comprising an expression vector of the 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 a 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.

Examples of mycobacteria for use in the present invention include Mycobacterium tuberculosis, mycobacterium bovis BCG (known as BCG), mycobacterium smegmatis (Mycobacterium smegmatis), mycobacterium avium (Mycobacterium avium complex) and other non-Mycobacterium tuberculosis (NTM). Examples of BCG strains useful 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 Czechoslokian), BCG Russia (also known as BCG Moscow), BCG Moreau (also known as BCG Brazil), BCG Japan (also known as BCG GTokyo), BCG Sweden (also known as Gothenburg), birkhaug, glaxo, BCG (also known as BCG Montreal), BCG Phipps, or other available strains.

Another embodiment of the present invention provides a method of treating diabetes comprising the steps of: administering to a subject having diabetes a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a protein that produces a STING agonist, or a functional portion thereof; and treating or preventing diabetes in the subject by providing a training immunity. Training immunity refers to the ability of one antigenic stimulus to elicit a stronger immune response against a second, different antigenic stimulus introduced at a later time. Training immunity is antigen-independent, based on heterologous CD4 and CD8 memory activation, cytokine-mediated, and is associated with epigenetic and metabolic changes. This approach results in the upregulation of glycolysis mediated by the training immunity. The upregulation of glycolysis mentioned above is beneficial for the prevention and treatment of type 1 and type 2 diabetes.

Another embodiment of the invention provides a method of stimulating a training immunity in a subject, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and stimulating a training immunity in the subject. An episomal change (episomal change) that upregulates glycolysis and/or stimulates histone methylation in a subject mediates a training immunity in the subject.

Another embodiment of the present invention provides a method of treating or preventing a viral infection in a subject, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and treating or preventing a viral infection in a subject. Stimulating a training immunity in a subject can treat or prevent a viral infection in the subject. Episomal changes that upregulate glycolysis in a subject and/or stimulate histone methylation in a subject mediate training immunity in the subject.

Another embodiment of the invention provides a method of treating or preventing a bacterial infection or a drug-resistant bacterial infection in a subject, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a protein that produces a STING agonist, or a functional portion thereof; and treating or preventing a bacterial infection or a drug-resistant bacterial infection in a subject. Stimulating a training immunity in a subject can treat or prevent a bacterial infection in the subject. An episomal alteration that upregulates glycolysis and/or stimulates histone methylation in a subject mediates a training immunity in the subject. The methods of the invention may employ one or more vectors of the invention or one or more bacterial strains comprising a vector of the invention.

Another embodiment of the invention provides a method of inhibiting 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 comprises the step of administering to a subject having a tumor a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a protein that produces a STING agonist or a functional portion thereof; inhibiting the expression of MDSC, M2 macrophages and Treg cells in the tumor; and inducing expression of macrophages, DCs and T effector cells in the tumor. Examples of M1 macrophages having inducible expression in tumors include M1 macrophages. Examples of T effector cells with inducible expression in tumors include CD4+ T cells and CD8+ T cells. Inhibition of expression of MDSCs, M2 macrophages and Treg cells was observed in tumors of subjects administered with mycobacteria comprising the vectors of the invention when compared to expression of MDSCs, M2 macrophages and Treg cells in tumors of reference subjects not administered with a pharmaceutical composition comprising a mycobacterium strain. Induction of expression of macrophages, DCs and T effector cells in a tumor was observed when compared to the expression of macrophages, DCs and T effector cells in a tumor of a reference subject that was not administered a pharmaceutical composition comprising a mycobacterium strain. Examples of suitable STING agonists 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' cGAMP and combinations thereof. Suitable vectors of the invention may comprise a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity Scan (DisA) protein or a functional portion thereof, and combinations thereof. By way of example, the tumor may be an epithelial cancer, breast cancer, or non-muscle invasive bladder cancer and melanoma. In some aspects, the tumor may be a non-muscle invasive bladder cancer, such as BCG non-responsive non-muscle invasive bladder cancer (BCG non-responsive NMIBC), and the pharmaceutical composition may be administered by intravesical instillation. In other aspects, the tumor can be a non-muscle invasive bladder cancer, such as BCG untreated non-muscle invasive bladder cancer (BCG untreated 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, bronchial cancer, and combinations thereof, and the pharmaceutical composition can be administered to the luminal surface of an epithelial cancer. In other aspects, the tumor is a solid tumor and the pharmaceutical composition is administered by intratumoral injection, intravenous injection, intradermal injection, transdermal injection, intravesical injection, topical injection, intramuscular injection, or subcutaneous injection. In other aspects, the tumor is a liquid tumor and the pharmaceutical composition is administered by intravenous injection, intradermal injection, transdermal injection, intravesical injection, topical injection, intramuscular injection, or subcutaneous injection. The methods of the 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), cimiralizumab (anti-PD-1 antibody), alemtuzumab (anti-PD-L1 antibody), avilimumab (anti-PD-L1 antibody), dewalimumab (anti-PD-L1 antibody), and combinations thereof.

Brief Description of Drawings

This application is a partial continuation of 16/638,943 (CIP), all figures from U.S. Ser. No. 16/638,943 are incorporated herein by reference in their entirety.

FIG. 1A-FIG. 1B from pSD5B P _hsp60 DISA plasmid constructs Mycobacterium that overexpress DISA release large amounts of c-di-AMP into the macrophage cytoplasm and transcribe high levels of DISA mRNA. FIG. 1A shows the use of a vector pSD5B P _hsp60 Mycobacterium tuberculosis of disA plasmid or wild type Mycobacterium tuberculosis (M.tb) (CDC 1551) J774 macrophages infected with an MOI of 1. The level of c-di-AMP in macrophages was determined by LC-MS/MS 24 hours after infection. As can be observed, the M.tb-disA-OE strain produced about 15 times more c-di-AMP than the wild-type Mycobacterium tuberculosis (CDC 1551). BCG-disA-OE is expected to show similarly high levels of c-di-AMP. (data from Dey B, dey RJ, cheung LS, pokkali S, guo H, lee JH, bishai WR. Abstract cyclic dinucleotides activities the cyclosalic surficial pathway and media administration to tuboculosis. Nat. Med.2015; 21-6. PMID. FIG. 1B shows a vector of pSD5B P _hsp60 BCG-Pasteur or BCG-Pasteur-WT of a disA plasmid grows to mid-exponential phase (mid-exponentatial phase). The bacteria were lysed and mRNA was prepared. Levels of disA mRNA were determined by quantitative RT-PCR. The BCG-disA-OE strain produces about 50 times more disA mRNA than BCG-Pasteur-WT.

FIG. 2. BCG overexpressing DISA increases proinflammatory cytokines. Gene expression profiling (gene expression profiling) of pro-inflammatory cytokines and IFN- β in mouse BMDM challenged with wild type BCG-Pasteur strain and DISA over-expressed BCG-Pasteur strain (qPCR).

FIG. 3. BCG over-expressing DISA increases IRF3 signaling. The effect of dis overexpression on the activation of the IRF pathway was measured by the IRF-SEAP QUANTI Blue reporter assay. Culture supernatants of infected RAW-Blue ISG cells were assayed for IRF activation. Images below the IRF activation map represent QUANTI Blue assay plates and sample wells; the processing parameters for a column of holes correspond to the parameters defined above for the columns aligned with the holes. The BCG-disA-OE in this figure is derived from BCG Pasteur.

FIGS. 4A-4C increase of proinflammatory cytokines in response to dis overexpression. FIG. 4A shows differential expression of TNF- α. FIG. 4B shows differential expression of IL-6. FIG. 4C shows differential expression of IL-1 β. Mouse BMDM was challenged with wild-type and dis A overexpressing strains of BCG-Pasteur. Culture supernatants were assayed for different cytokines by ELISA.

FIG. 5. BCG overexpressing DISA induces a differential immune response in human bladder cancer cells (RT 4). Differential gene expression in human RT4 bladder cancer cells challenged with wild-type BCG-Pasteur, wild-type BCG-Tice strain, and BCG-Pasteur-dis A-OE. Quantitative real-time PCR based on SYBR green was used to measure mRNA expression levels.

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

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

Figure 8. Analysis of the immune spectrum of MNU-induced Fischer rat bladder tumors in response to intravesical therapy with different BCG strains. Differential gene expression in rat bladder tumor cells following therapy with wild-type mycobacterium bovis BCG-Pasteur strain and mycobacterium bovis BCG-Pasteur strain with dis a overexpression. The expression level of mRNA was measured using TaqMan-based quantitative real-time PCR. BCG-WT is BCG Pasteur and BCG-disA-OE is derived from BCG Pasteur.

FIG. 9 Gene expression profiling of bladder from untreated MNU tumor bearing rats (MNU tumor bearing rats) or MNU tumor bearing rats treated with WT or dibG overexpressing disA.

FIG. 10. Summary of relative gene expression by BCG-disA-OE and 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 for 8 weeks by intravesical instillation and then treated with BCG-disA-OE or BCG-WT for 8 weeks by intravesical instillation. At week 16 necropsy, bladders were removed and mRNA prepared. Quantitative RT-PCR was performed on the indicated cytokine or chemokine genes. The changes shown are the fold induction or reduction of observed BCG-disA-OE normalized against the observed BCG-WT. BCG-WT is BCG Pasteur and BCG-disA-OE is derived from BCG Pasteur.

Figure 11. Two cyclic dinucleotide cyclases and phosphodiesterase proteins present in bcg: diagrams of 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.

FIG. 12. Carrying pSD5BP compared to wild type Mycobacterium tuberculosis (Mtb-CDC 1551) _hsp60 Mycobacterium tuberculosis (M.tb-dis A-OE or Mtb-OE) of a dis plasmid is significantly attenuated in mice. Female BALB/c mice (n = 10/group) 6-7 weeks old were infected with-3.5 log10 CFU by aerosol infection (aerosol infection) as described above. On day 1, 3 mice in each group were counted for CFU and confirmed to have implanted 3.5log10 CFU units. Mice remained housed until death. As can be observed, the median time to death for wild-type mycobacterium tuberculosis infection was 150.5 days. In contrast, mice infected with the same M.tb-disA-OE (Mtb-OE) inoculum hadMedian death time (p) of 321.5 days<0.001). Compared to BCG-WT, BCG-disA-OE is expected to show a similar loss of virulence in mice. (data from Dey B, dey RJ, cheung LS, pokkali S, guo H, lee JH, and Bishai WR. A bacterial cyclic pyridine activities the cyclosalic surficial path and media existence resistance to tuboculosis. Nat. Med. (2015); 21-6. PMID 25730264).

Fig. 13A-fig. 13B other BCG strains were also active: the BCG Tice strain overexpressing DISA also showed similar proinflammatory cytokine induction as the BCG Pasteur overexpressing DISA. Bone marrow-derived macrophages were challenged with wild type strains of both BCG Pasteur and BCG tie strains and with a strain overexpressing disA at an m.o.i. of 1. Culture supernatants were harvested and cytokines were detected using ELISA. FIG. 13A shows the differential expression pattern of TNF- α. FIG. 13B shows differential expression of IL-6. Mouse BMDM was challenged with two different BCG strains. The BCG-Tice strain is from a commercially available Onco-Tice product.

FIG. 14 type I interferon response in macrophages in response to BCG-disA-OE is STING dependent. Bone marrow-derived macrophages from STING ablated (KO) and control mice were challenged with wild-type and disA OE strains of BCG Pasteur for 24h. The culture supernatants were probed for IFN- β levels using ELISA.

Figure 15 shows that intravesical instillation of BCG-disA-OE showed maximal antitumor efficacy (statistically significant pathological improvement) in the MNU oncogenic model of non-muscle invasive bladder cancer (NIMBC). Within the first 8 weeks, the rat groups received 4 intravesical treatments with MNUs (one treatment every 2 weeks) to elicit NIMBC. Over the next 8 weeks, rats received 4 intravesical treatments (one treatment every 2 weeks) with PBS (untreated), BCG-WT, or BCG-DISA-OE. At the end of the 16-week experiment, the rats were sacrificed and their bladders removed. A portion of the bladder was fixed and H & E stained and then interpreted blindly by a committee-certified urologist. Tumor involvement scores and cancer stages (T2-3, T1, CIS + papillary lesions, CIS alone or normal-dysplasia) were determined and shown. As can be observed, BCG-disA-OE instillation resulted in a statistically significantly lower tumor involvement index than PBS (untreated), whereas BCG-WT was not statistically significantly superior to PBS. This 16 week experiment was performed twice. The data in fig. 7 represent the results of experiment 1. The data in this figure (fig. 15) represents the combined results of experiment 1 plus experiment 2. The qPCR data shown in figures 8 and 9 were obtained from the use of bladder tissue at necropsy at the end of experiment 1.

FIG. 16 shows that BCG-disA-OE reduces Treg (CD 4) in syngeneic (syngeneic) bladder cancer tumors in mice ⁺ CD25 ⁺ Foxp3 ⁺ ). Mice were implanted 5X10 ventral ⁶ And BBN975 mouse bladder cancer tumor cells. When the tumor diameter was 1.5cm, mice received 3 intratumoral injections of PBS (control), BCG-WT or BCG-disA-OE (treatment every 2 days). Two days after the last intratumoral treatment, mice were sacrificed and their spleens and tumors were removed. After tumor cells were dispersed, the cell preparations were stained and flow cytometry was performed. As can be observed, BCG-disA-OE results in reduced tumor CD4 ⁺ Treg, reduced tumor CD8 ⁺ Treg and reduced splenic CD4 ⁺ Treg。

FIGS. 17A-17B show that BCG-disA-OE is safer than BCG-WT in both mouse models. FIG. 17A shows the exposure of a group of BALB/c mice (immunocompetent) to 1X 10 using a Glas-Col nebulizing chamber ³ BCG-WT or BCG-disA-OE of CFU (confirmed by sacrifice of a group of mice and determination of lung CFU count on day 1). After 4 weeks, mice from each group were sacrificed, their lungs removed, homogenized, and plated on 7H11 agar plates. The graph shows the mean CFU counts of the lungs of BCG-WT and BCG-disA-OE infected mice. As can be observed, a statistically significantly lower lung CFU load of BCG-disA-OE compared to BCG-WT was observed. FIG. 17B shows the exposure of SCID mouse (immunosuppressed) groups to 1X 10 using a Glas-Col nebulization chamber ² BCG-WT or BCG-disA-OE of CFU (confirmed by sacrifice of a group of mice and determination of lung CFU count on day 1). The third group was not infected. The graph shows the Kaplan-Meier survival curves for the mouse groups. As can be observed, BCG-disA-OE infected mice had statistically greater susceptibility than BCG-WT infected miceSignificantly longer survival time.

FIG. 18 shows a CD14 ⁺ In human monocytes, BCG-disA-OE elicits statistically significantly higher levels of "Trained immunological and epigenetic markers" (Trained immunological and epigenetic marks) than BCG-WT. By "training immunity" is meant the ability of a first immunostimulatory substance to induce an enhanced immune response to a subsequently administered second, differently antigenic stimulatory substance. In this experiment, CD14 ⁺ Human monocytes were prepared from LeukoPak collected by apheresis. On day 0, CD14 was removed ⁺ Human monocytes were infected with BCG-WT or BCG-disA-OE at an MOI of 5:1 for 3 hours. The third group of cells was not infected. After infection, cells were washed more than once (every two days). After a resting time (rest period) of 6 days, monocytes were restimulated with the TLR1/2 agonist PAM3CSK4 for 2 hours. The cells were washed repeatedly and then incubated for 24h. The level of secreted IL-1. Beta. In the culture supernatants was measured by ELISA. As can be observed, while BCG-WT by itself elicits a statistically significantly higher level of immune response against the second stimulus, BCG-disA-OE elicits a statistically significantly higher response than either BCG-WT or uninfected cells, as compared to uninfected cells.

FIG. 19 shows that BCG-disA-OE elicits a larger histone activation signature (H3K 4-trimethylation) in the promoter regions of the IL6 and TNF genes than BCG-WT. "training immunity" refers to the ability of a first immune stimulant to induce an enhanced immune response against a subsequently administered second, different antigenic stimulant. Training immunity is associated with epigenetic modifications such as histone methylation in the promoter regions of cytokines and other immune mediators. The experiment shown in figure 19 was performed in the same cell group and in exactly the same manner as described in figure 18, except that: following a second stimulation with the TLR1/2 agonist PAM3CSK4 (abbreviated PAM 3), the fixed cells were harvested, chromatin cross-linked, and DNA was collected for chromatin immunoprecipitation analysis (ChIP) using antibodies specific for the H3K4-me3 histone methylation marker. H3K4-me3 is known as a gene activation marker. The figure shows the relative fold change in abundance of immunoprecipitated DNA as measured by quantitative PCR using primers for the IL6 and TNF gene promoter regions. As can be observed, both BCG-Pasteur-disA-OE and BCG-Tice-disA-OE resulted in significantly greater levels of trimethylation of the H3K4 histone in the IL6 and TNF promoter regions than their corresponding BCG-WT strains, following challenge with the second stimulus PAM3CSK4.

FIG. 20 shows the successful construction of BCG-Tice-dis-A-OE. Previous work by the present inventors has utilized BCG-Pasteur to construct BCG-Pasteur-dis A-OE. This strain was provided by Frank Collins, doctor 1995 to one of the present inventors. This strain is the same strain known as BCG-Pasteur-Aeras. BCG-Tice is manufactured and sold by Merck and is the only FDA approved BCG available in the United states. The present inventors purchased BCG-Tice, prepared electrocompetent BCG-Tice, and electroporated the pSD5-hsp60-MT3692 plasmid into BCG-Tice. The figure shows the results of colony PCR of 5 kanamycin-resistant candidate clones of transformed BCG-Tice, and confirms that BCG-Tice-DISA-OE was successfully prepared by electroporation of pSD5-hsp65-MT3692 plasmid into BCG-Tice. Note the nomenclature, the present inventors previously referred to this same plasmid pSD5-hsp60-MT3692. However, the actual promoter in this strain is the promoter of the hsp65 gene of mycobacterium leprae (m.leprae). Thus, the present inventors now more correctly named the plasmid pSD5-hsp65-MT3692.

FIG. 21 shows clone 2 of BCG-Tice-DISA-OE from the transformation experiment shown in FIG. 20 strongly expresses the DISA gene. Real-time PCR was used to show differential dis A expression in four different BCG-Tice-dis-OE clones. Gene expression was measured in total RNA isolated from log late cultures using log phase cultures using SYBR green based quantitative real-time PCR. The data points illustrated represent the mean ± standard error of the mean (SEM) of 3 independent experiments. Mycobacterium tuberculosis sigA (Rv 2703) was used as an internal control. Use 2 ^-ΔΔCT The method performs data analysis. Welch correction (. About.P) after Student's t-test<0.001；**P<0.01). The inventors generated a seed lot (seed lot) of BCG-Tice-disA-OE clone 2, and this clone was abbreviated as“BCG-Tice-disA-OE”。

FIG. 22 shows potent, statistically significantly enhanced induction of IRF3 in mouse bone marrow-derived macrophages infected with BCG-Pasteur-disA-OE compared to BCG-Pasteur-WT. Mouse (C57 BL/6) bone marrow derived macrophages were infected with wild type BCG Pasteur and a strain overexpressing DISA (20 MOI) for 3h. Cells were washed with warm DPBS to remove non-internalized bacilli (bacillus) and then incubated for another 3 hours. IRF3 expression was measured in total RNA isolated from cell lysates using SYBR green based quantitative real-time PCR. The data points illustrated represent the mean ± standard error of the mean (SEM) of 3 independent experiments. Mouse β -actin was used as an internal control. Use 2 ^-ΔΔCT The method performs data analysis. Welch correction (. About.P) after Student's t-test<0.001；**P<0.01)。

FIG. 23 shows that STING is required for type I IFN (IFN-. Beta.) induction in response to BCG-WT and BCG-disA-OE enhancement. Mice from wild-type animals with STING ablation (STING-KO) (C57 BL/6) bone marrow-derived macrophages were infected with different BCG strains (MOI =1 20) for 3h. Cells were washed with warm DPBS to remove non-internalized bacilli and then incubated for an additional 24h before harvesting the culture supernatant. ELISA for IFN- β was performed in culture supernatants as per the manufacturer's instructions. Data points represent the mean ± standard error of the mean (s.e.m.) of 3 independent biological experiments. The Student's t-test was followed by Welch correction (. P < 0.01).

FIGS. 24A-24C show that interferon- β is induced in murine BMDM, BMDC, and J774.1 macrophages when exposed to strain BCG that overexpresses DISA, and that IFN- β responses to BCG-Pasteur-DISA-OE and BCG-Tie-DISA-OE are statistically significantly greater than the corresponding BCG-WT strains. Mice (C57 BL/6) Bone Marrow Derived Macrophages (BMDM) and J774.1 macrophages were infected with different BCG strains (MOI: 20) for 3h. The non-internalized bacilli were washed with warm DPBS and the cells were incubated for an additional 24 hours. IFN- β levels in culture supernatants were quantified using ELISA according to the manufacturer's instructions. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. About.p < 0.001;. About.p < 0.01;. About.p < 0.05). Figure 24A shows interferon- β levels in murine BMDM. Figure 24B shows interferon- β levels in murine BMDCs. Figure 24C shows interferon- β levels in murine J774.1 macrophages.

FIGS. 25A-25C show that IL-6 was induced in mouse BMDM, BMDC, and J774.1 macrophages in response to exposure to a BCG strain that is over-expressed by DISA, and that the IL-6 response to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE is statistically significantly greater than the corresponding BCG-WT strains. Mice (C57 BL/6) Bone Marrow Derived Macrophages (BMDM) and J774.1 macrophages were infected with different BCG strains (MOI: 20) for 3h. The non-internalized bacilli were washed with warm DPBS and the cells were incubated for an additional 24 hours. The IL-6 level in the culture supernatants was quantified using ELISA according to the manufacturer's instructions. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. About.p < 0.001;. About.p < 0.01;. About.p < 0.05). FIG. 25A shows IL-6 levels in murine BMDM. FIG. 25B shows IL-6 levels in murine BMDC. FIG. 25C shows IL-6 levels in murine J774.1 macrophages.

FIGS. 26A-26C show that TNF was induced in mouse BMDM, BMDC, and J774.1 macrophages in response to exposure to strains of BCG that are overexpressed by DISA, and that the response to BCG-Pasteur-DISA-OE and BCG-tip-DISA-OE was statistically significantly greater than the corresponding BCG-WT strains. Mice (C57 BL/6) Bone Marrow Derived Macrophages (BMDM) and J774.1 macrophages were infected with different BCG strains (MOI: 20) for 3h. The non-internalized bacilli were washed with warm DPBS and the cells were incubated for an additional 24 hours. TNF levels in culture supernatants were quantified using ELISA according to the manufacturer's instructions. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (.;. P < 0.001;. P < 0.01;. P < 0.05). Figure 26A shows TNF levels in murine BMDM. Figure 26B shows TNF levels in murine BMDCs. Figure 26C shows TNF levels in murine J774.1 macrophages.

FIGS. 27A-27B show that TNF and IFN- γ were induced in the rat bladder cancer NBT-II cell line in response to exposure to a BCG strain that is overexpressed by disA, and that both responses to BCG-Pasteur-disA-OE and BCG-Tie-disA-OE were statistically significantly greater than the corresponding BCG-WT strains. NBT-II cells were infected with wild type and recombinant strains of BCG for 3h. The non-internalized bacilli were washed repeatedly with warm DPBS and the cells were incubated for an additional 24h. Culture supernatants were used to quantify TNF and IFN-. Gamma.. Data points represent 3 independent biological experiments ± standard error of 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- γ levels in NBT-II cells.

FIGS. 28A-28D show that IFN- β, IFN- γ, TNF, and IL-1 β were induced in human transitional cell papilloma RT4 bladder cancer cell line in response to exposure to a BCG strain that is overexpressed by dis A, and that both responses to BCG-Pasteur-dis-A-OE and BCG-tip-dis-A-OE were greater than the corresponding BCG-WT strains. RT4 cells were infected with wild type and recombinant strains of BCG for 3h. The non-internalized bacilli were washed repeatedly with warm DPBS and the cells were incubated for an additional 24h. Culture supernatants were used for the quantification of cytokines according to the manufacturer's instructions. Data points represent 2 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. About.p < 0.001;. About.p < 0.01;. About.p < 0.05). FIG. 28A shows IFN- β levels in RT4 cells. Figure 28B shows IFN- γ levels in RT4 cells. Figure 28C shows TNF levels in RT4 cells. FIG. 28D shows IL-1 β levels in RT4 cells.

FIG. 29 shows that BCG-disA-OE stimulates increased IFN- β levels in more than one bladder cancer cell line to a greater extent than BCG-WT. The graph shows the level of IFN- β mRNA (by 2) after exposure to BCG-WT, BCG-disA-OE and LPS ^-ΔΔCT Relative expression of the method). 5637 cells are human muscle-layer invasive bladder cancer cells, RT4 cells are human transitional cell papilloma bladder cancer cells, and NBT-II cells are rat bladder cancer cells induced by N-butyl-N- (-4-hydroxybutyl) nitrosamine.

FIGS. 30A-30D show cytokine responses of IFN- β, IFN- γ, IL-6 and TNF in the lungs of BCG-WT and BCG-DISA-OE infected mice at different time points following aerosol infection. The figure reveals that the response to BCG-Pasteur-disc-OE and BCG-Tice-disc-OE is greater than for the corresponding BCG-WT strain at most time points for most cytokines. BALB/c mice were infected by aerosol route as described in FIG. 19. Groups of mice were sacrificed at 2,4 and 6 weeks post infection. Lung homogenates were prepared and cytokine levels were quantified using ELISA according to the manufacturer's protocol (n =4 animals/treatment group ± s.e.m.). Data analysis was performed using paired t-tests (. About.p < 0.001;. About.p < 0.01;. About.p < 0.05). FIG. 30A shows the levels of IFN- β in the lungs of BCG-WT and BCG-disA-OE infected mice. FIG. 30B shows IFN- γ levels in the lungs of BCG-WT and BCG-disA-OE infected mice. FIG. 30C shows IL-6 levels in the lungs of BCG-WT and BCG-disA-OE infected mice. FIG. 30D shows TNF levels in the lungs of BCG-WT and BCG-disA-OE infected mice.

FIGS. 31A-31D show cytokine responses of IFN- β, IFN- γ, IL-6 and TNF in the spleens of BCG-WT and BCG-disA-OE infected mice 4 weeks after aerosol infection. The figure reveals that for most cytokines, the response to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE is greater than for the corresponding BCG-WT strains. BALB/c mice were infected by aerosol route as depicted in FIG. 17. Groups of mice were sacrificed 4 weeks post infection. Spleen homogenates were prepared and cytokine levels were quantified using ELISA according to the manufacturer's protocol (n =4 animals/treatment group ± s.e.m.). Data analysis was performed using paired t-tests (. About.p < 0.001;. About.p < 0.01;. About.p < 0.05). FIG. 31A shows the levels of IFN- β in the spleen of BCG-WT and BCG-disA-OE infected mice. FIG. 31B shows IFN- γ levels in the spleen of BCG-WT and BCG-disA-OE infected mice. FIG. 31C shows IL-6 levels in the spleen of BCG-WT and BCG-disA-OE infected mice. FIG. 31D shows TNF levels in the spleen of BCG-WT and BCG-disA-OE infected mice.

FIG. 32 shows the strategy used to generate "pSD5. HspP 65-disA. PanCD- -Kan-free" (SEQ ID NO: 31). This protocol replaces the Kan cassette "pSD5.Hsp65-dis A. Kan" (SEQ ID NO: 30) with the panCD operon to produce "pSD5.Hsp65-dis A. PanCD- -without Kan" (SEQ ID NO: 31).

FIG. 33 shows the molecular structure of pJV53, which is a recombinant engineered plasmid of SEQ ID NO. 32.

FIG. 34 shows the molecular structure of pUC-Hyg, a plasmid with the Hyg cassette flanked by dif sites, as SEQ ID NO 35.pUC-Hyg was used to generate the plasmid "pUC-Hyg-panCD-KO" (SEQ ID NO: 36).

FIG. 35 shows the molecular structure of the plasmid "pUC-Hyg-panCD-KO" as SEQ ID NO: 36. "pUC-Hyg-panCD-KO" was produced by cloning 500bp of the panCD 5'UTR on one flank of the Hyg cassette and 500bp of the panCD 3' UTR on the other flank.

FIG. 36 shows the molecular structure of plasmid "pSD5.Hsp65-dis A. Kan" as SEQ ID NO: 30.

FIG. 37 shows the molecular structure of the plasmid "pSD5.Hsp65-dis A. PanCD-Kan-free" SEQ ID NO: 31. This plasmid was generated using the protocol illustrated in figure 32.

Fig. 38 shows the number of positive samples.

FIGS. 39A-39C show confirmation of the M.tb-disA overexpression phenotype and induction of IRF signaling of BCG-disA-OE. FIG. 39A shows colony PCR using kanamycin gene-specific primers to confirm the presence of recombinant plasmid pSD5-hsp60-MT3692 in BCG-disA-OE (choice) clones selected for kanamycin (25. Mu.g/mL). FIG. 39B shows real-time PCR showing differential disA expression in different cloned BCG Tice, BCG-disA-OE Tice. Transcript levels in total RNA isolated from log late phase cultures were measured using log phase cultures. Mycobacterium tuberculosis sigA (Rv 2703) is used as a reference gene and passes 2 ^ΔΔCT The method calculates the relative expression. Fig. 39C shows measurement of IRF activation by quantification of IRF induction based on ISRE (RLU, relative light units) in culture supernatants 24h after infection of RAW-Lucia ISG cells (MOI = 1. Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

FIGS. 40A-40C show that BCG strains overexpressing C-di-AMP are potent inducers of type I interferon in a STING-dependent manner. FIG. 40A shows quantitative measurements of IFN- β in culture supernatants of wild-type C57 BL/6-derived BMDM and STING-KO BMDM (C57 BL/6J-Tmem173 gt/J) 24h post BCG-disA-OE (Tice) infection. FIG. 40B shows quantitative measurements of IFN- β in culture supernatants of wild-type C57 BL/6-derived BMDM, BMDC, J774.1 macrophages and human monocyte-derived macrophages (HMDM) 24h post infection. FIG. 40C shows quantitative measurements of IFN- β in culture supernatants of wild-type C57 BL/6-derived BMDM, BMDC, J774.1 macrophages, and human monocyte-derived macrophages (HMDM) 24h post infection. Macrophage to BCG infection ratio = 1. Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIGS. 41A-41D show that BCG strains overexpressing c-di-AMP are potent inducers of proinflammatory cytokines, TNF- α, and IL-6. (A-B) quantitative measurement of TNF- α in culture supernatants of wild type C57BL/6 derived BMDM, BMDC, J774.1 macrophages and Human Monocyte Derived Macrophages (HMDM). FIG. 41A shows measurements 24h after infection with BCG-disA-OE (Tice). FIG. 41B shows measurements 24h after infection with BCG-disA-OE (Pasteur). (C-D) quantitative measurement of IL-6 in culture supernatants of wild-type C57 BL/6-derived BMDM, BMDC, J774.1 macrophages and human monocyte-derived macrophages (HMDM). FIG. 41C shows measurements 24h after infection with BCG-disA-OE (Tice). FIG. 41D shows measurements 24h after infection with BCG-disA-OE (Pasteur). Macrophage to BCG infection ratio = 1. Data represent mean ± SEM (n =3 replicates). . Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIG. 42 shows that BCG overexpressing c-di-AMP strongly induces the proinflammatory cytokine TNF- α in a STING-dependent manner. Quantitative measurement of TNF- α in culture supernatants of wild-type C57 BL/6-derived BMDM and STING-KO BMDM (C57 BL/6J-Tmem173 gt/J) 24h after BCG-disA-OE (Tice) infection. Macrophage to BCG infection ratio = 1. Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIGS. 43A-43E show that BCG-disA-OE induced significantly higher Th1 cytokines and chemokines compared to WT BCG.FIG. 43A shows relative gene expression analysis of different cytokines and chemokines in IFN- γ activated macrophages 6h after infection with wild type BCG (Tice) and BCG-dis A-OE (Tice) strains. (B-D) analysis of the relative gene expression of IL-6, IL-12 and MCP-1 in IFN-. Gamma.activated macrophages 6h after infection with wild-type BCG (Pasteur) and BCG-disA-OE (Pasteur) strains. Beta-actin was used as a reference gene, and was passed through 2 ^ΔΔCT The method calculates the relative expression. 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 the quantitative measurement of MCP-1 in culture supernatants of wild-type C57 BL/6-derived BMDM 24h post BCG-disA-OE (Tice) infection. Macrophage to BCG infection ratio = 1. Data represent mean ± SEM (n =3 replicates). Student's t test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

Fig. 44A-44C show differential apoptosis induction in murine BMDM and J774.1 macrophages after infection with different BCG strains. (A-B) murine BMDM and J774.1 macrophages were challenged with BCG WT or BCG-DISA-OE strain at a MOI of 1. Representative data from an individual infection assay. Fig. 44A shows measurements in murine BMDM. Fig. 44B shows measurements in J774.1 macrophages. Fig. 44C shows a bar graph showing quantification of late apoptotic cell death following infection. Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P <0.05.

FIGS. 45A-45C show internalization and differential toxicity of WT and BCG-disA-OE strains in human urothelial cancer 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 carcinoma) cells of wild-type BCG exposed to different MOIs. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay. Figure 45A shows cell viability of RT4 cells. Figure 45B shows cell viability of 5637 cells. Fig. 45C shows cell viability of J82 cells.

FIGS. 46A-46D show that BCG Tice overexpressing c-di-AMP is a stronger inducer of anti-tumor cytokine responses in urothelial cancer cells. (A-D) differential TNF-. Alpha.IL-6, IL-1. Beta. And IFN-. Gamma.levels were quantified using ELISA 24h after infection with different wild-type BCG (Tice) and BCG-disA-OE (Tice) strains in different urothelial cancer cells. Cells were infected at a ratio to BCG =1, and data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001. FIG. 46A shows TNF- α levels. FIG. 46B shows IL-6 levels. FIG. 46C shows IL-1 β levels. Figure 46D shows IFN- γ levels.

FIGS. 47A-47G show that BCG Pasteur overexpressing c-di-AMP is a stronger inducer of anti-tumor cytokine responses in urothelial cancer cells. (A-G) differential cytokine levels in different urothelial cancer cells were quantified using ELISA 24h after infection with different wild-type BCG (Pasteur) and BCG-disA-OE (Pasteur) strains. Cell to BCG infection ratio = 1. Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001. FIG. 47A shows TNF- α levels in 5637 cells. Figure 47B shows TNF-a levels in BBN975 cells. FIG. 47C shows IL-6 levels in UPPL1595 cells. FIG. 47D shows IL-1 β levels in MB49 cells. FIG. 47E shows IL-1 β levels in UPPL1595 cells. Figure 47F shows IFN- γ levels in BBN975 cells. FIG. 47G shows IFN- γ levels in NBT-II cells.

FIGS. 48A-48B show the reprogramming of stronger macrophages to the M1 phenotype following infection with a BCG strain overexpressing c-di-AMP. Figure 48A shows wild-type BMDM infected with different BCG strains and infected at an MOI of 1. Cell surface and intracellular staining were performed and cells were analyzed using flow cytometry (BD LSR II flow cytometer). Showing TNF-alpha positive antigen presenting mouse macrophages (MHC Class II) after infection with wild type BCG Tice and Pasteur strains and BCG Tice and Pasteur strains overexpressing c-di-AMP ⁺ CD11b ⁺ F4/80 ⁺ ) Histogram of percentage of (c).The data was processed using FlowJo software (Tree Star v 10). Figure 48B shows a representative flow diagram showing different cell phenotypes of antigen-producing M1 macrophages. Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

Fig. 49A-49B show the reprogramming of stronger M2 macrophages after infection with BCG strains overexpressing c-di-AMP. FIG. 49A shows mouse BMDM macrophages (CD 11 b) following infection with wild type ⁺ F4/80 ⁺ ) Upper M2 macrophage surface marker (CD 206) ⁺ CD124 ⁺ ) Percentage of positive. FIG. 49B shows M2 macrophages (CD 206) ⁺ CD124 ⁺ Mouse macrophages) population of mice). Briefly, wild-type BMDM was produced in the presence of murine M-CSF. Macrophages were infected with different BCG strains and infected at an MOI of 1. Cell surface and intracellular staining were performed and cells were analyzed using flow cytometry (BD LSR II flow cytometer). Data were processed using FlowJo software (Tree Star v 10). Data represent mean ± SEM (n =3 replicates). Student's t test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

FIGS. 50A-50B show a stronger induction of IL-10 secreting monocytic MDSCs in murine BMDM following infection with wild type BCG strain and BCG strain overexpressing c-di-AMP. FIG. 50A shows the percentage of M-MDSC in total myeloid cells (CD 45 +). FIG. 50B shows the percentage of M-MDSCs that produce IL-10 after infection of murine BMDM with WT and BCG-disA-OE strains. Briefly, wild-type BMDM macrophages were infected with different BCG strains and infected at an MOI of 1. Cell surface and intracellular staining were performed and cells were analyzed using flow cytometry (BD LSR II flow cytometer). Data were processed using FlowJo software (Tree Star v 10). Data represent mean ± SEM (n =3 replicates). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIGS. 51A-51B show differential induction of classical (inflammatory) monocytes after infection with wild-type BCG Tice and BCG Tice overexpressing c-di-AMP. FIG. 51A showsDisplay of classical monocytes (CD 14) in the CD11b population ⁺ CD16 ^- ) Histogram of percentage (d). Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors. Negatively selected human monocytes were infected with the wild type strain and the BCG-disA-OE strain for 24h (MOI of 1. Fig. 51B shows a representative flow cytometry plot showing different percentages of monocyte populations. Cell surface staining was performed and cells were analyzed using flow cytometry (BD LSR II flow cytometer). The data was processed using FlowJo software (Tree Star v 10). Student's t-test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

FIGS. 52A-52B show potent inducers of pro-inflammatory cytokines in human monocyte-derived macrophages with BCG overexpressing c-di-AMP. FIG. 52A is a graph showing the production of TNF- α (TNF- α) ⁺ HLA-DR ⁺ /CD14 ⁺ CD16 ^- ) And IL-6 (IL-6) ⁺ HLA-DR ⁺ /CD14 ⁺ CD16 ^- ) Histogram of the percentage of MHC class II positive classical macrophages. Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors. Negatively selected human monocytes are allowed to differentiate into macrophages. Macrophages were infected with the wild type strain and BCG-disA-OE strain for 24h (MOI of 1. Fig. 52B shows a representative flow cytometry plot showing different percentages of macrophage populations. Cell surface staining was performed and cells were analyzed using flow cytometry (BD LSR II flow cytometer). Data were processed using FlowJo software (Tree Star v 10). Student's t-test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

FIGS. 53A-53C show that BCG overexpressing C-di-AMP strongly inhibited the M2 macrophage phenotype. Figure 53A shows the percentage of immunosuppressive M2 macrophages (CD 206+ CD163 +) out of total transitional macrophages (CD 14+ CD16 +). FIG. 53B shows the percentage of IL-10 producing macrophages in total M2 macrophages (CD 206+ CD163 +) following infection of HMDM with the WT and BCG-disA-OE Tice strains. FIG. 53C shows a representative flow cytometry plot showing the M2 cell surface phenotype of M2 macrophages and IL-10 producing cells. Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors. Negatively selected human monocytes are allowed to differentiate into M2 macrophages in the presence of M-CSF. Infection was performed for 24h (MOI of 1. Data are mean ± SEM (n =3 replicates on monocyte-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 shows that phagocytosis is enhanced by BCG-infected macrophages overexpressing c-di-AMP. HMDM was infected with WT BCG strain and BCG-dis-A-OE strain for 6h and phagocytic activity was measured by quantification of intracellular FITC labeled IgG opsonized latex beads. Images were collected on live cells. Nuclear staining was performed using Hoechst. Image acquisition was performed using an LSM700 confocal microscope at 63X magnification. Images were processed using Fiji software. Cell to BCG infection ratio = 1. Data are mean ± SEM (n =3 replicates on monocyte-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 fluorescence quantification.

FIGS. 55A-55B show that BCG overexpressing c-di-AMP is a potent inducer of pro-inflammatory cytokines in primary human monocytes. (A-B) BCG-disA-OE induced significantly higher TNF- α and IL-6 gene expression in primary human monocytes compared to WT BCG. TNF-a and IL-6 expression was obtained in primary human monocytes isolated from different healthy donors using qRT-PCR. RNU6A was used as reference gene and was crossed by 2 ^ΔΔCT The method calculates the relative expression. Data represent mean ± SEM (n =6 different healthy donors). Student's t test (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001. FIG. 55A shows TNF- α levels. FIG. 55B shows IL-6 levels.

FIGS. 56A-56E show that BCG overexpressing c-di-AMP is a stronger inducer of the training immune epigenetic marker in trained human monocytes. (A-B) shows a bar graph of epigenetically active chromatin marker H3K4me3 on the TNF-. Alpha.and IL-6 gene promoters. BCG-trained human monocytes isolated from different donors (n =4 different healthy donors) were enriched for the epigenetically modified TNF-a and IL-6 gene promoters after restimulation with Pam3CSK4. The H3K 4-trimethylated promoter was enriched and quantified using ChIP-PCR. FIG. 56A shows H3K4me3 levels on the TNF- α gene promoter. FIG. 56B shows the H3K4me3 levels on the IL-6 gene promoter. (C-D) shows a bar graph of the epigenetically inactivated chromatin marker H3K9me3 on the TNF-. Alpha.and IL-6 gene promoters. BCG-trained human monocytes isolated from different donors (n =4 different healthy donors) were enriched for the epigenetically modified TNF-a and IL-6 gene promoters after restimulation with Pam3CSK4. The H3K 9-trimethylated promoter was enriched and quantified using ChIP-PCR. Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001. FIG. 56C shows H3K9me3 levels on the TNF- α gene promoter. FIG. 56D shows H3K9me3 levels on the IL-6 gene promoter. Figure 56E shows a schematic of ex vivo BCG training.

FIGS. 57A-57C show that BCG overexpressing C-di-AMP exhibits improved anti-tumor activity in the MNU carcinogen model of NMIBC. Figure 57A schematic representation of the intravesical treatment strategy of BCG in the MNU carcinogen model of NMIBC. Fig. 57B is a histogram showing tumor involvement index. Fig. 57C is a graph showing tumor stage. Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIGS. 58A-58C show BCG-disA-OE strains with attenuated virulence in vivo. FIG. 58A illustrates a BALB/c BCG aerosol challenge model. FIG. 58B shows BALB/c mouse lung bacterial load of wild type strain and BCG-disA-OE strain in mouse lungs 4 weeks after infection. Data are mean ± s.e.m. (n =5 animals/group). FIG. 58C shows the engraftment of BCG strains following aerosol challenge in BALB/C mice (day 01). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIGS. 59A-59C show attenuated C-di-AMP overexpressing BCG strains in a severe immune compromised (SCID) mouse model of aerosol infection. Figure 59A illustrates a SCID mouse model of BCG aerosol infection. Fig. 59B shows the survival of SCID mice (n = 10) after infection with different BCG strains. Figure 59C shows the engraftment of BCG strains following aerosol challenge in SCID mice (day 01). Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIG. 60A-FIG. 60D show that BCG overexpressing c-di-AMP is a stronger inducer of Th1 cytokines in vivo. (A-B) quantification of IFN- β, TNF- α, IL-6 and IFN- γ in lung homogenates from BALB/c mice 4 weeks after infection with different BCG strains using ELISA. FIG. 60A shows the results of WT BCG (Rice) and BCG-DISA-OE (Rice). FIG. 60B shows the results for WT BCG (Pasteur) and BCG-disA-OE (Pasteur). (C-D) quantification levels of IFN- β, TNF- α, IL-6 and IFN- γ in lung homogenates from BALB/C mice 4 weeks after infection with different BCG strains using ELISA. FIG. 60C shows the results for WT BCG (Tice) and BCG-disA-OE (Tice). FIG. 60D shows the results of WT BCG (Pasteur) and BCG-disA-OE (Pasteur). Results are expressed as mean (pg/ml) ± SEM (n =4 animals/group). Student's t test. Student's t-test (double tail) P <0.05,. P <0.01,. P <0.001,. P <0.0001.

FIGS. 61A-61B show that therapeutic intratumoral injection of BCG overexpressing c-di-AMP resulted in greater antitumor activity in the MB49 model of bladder cancer. Fig. 61A shows a schematic of an intratumoral injection of a tumor. Mice were implanted at 1X 10 on day 0 ⁵ Individual MB49 cells, and tumor volumes were then obtained until the group averaged-40 mm ³ . At this time, every 3 days in a total volume of 50. Mu.L with 5X10 ⁶ Wild type or BCG-disA-OE strain or mice treated with PBS alone for 3 treatments. FIG. 61B shows tumor growth in animals bearing MB49 after treatment as shown in FIG. 61A, treated with vehicle (PBS), wild type and BCG-disA-OE. Two-way ANOVA.

FIGS. 62A-62D show that BCG overexpressing c-di-AMP induced stronger IFN- γ infiltration at the tumor site following intratumoral administration. (A-B) dot plots showing the relative abundance percentage of CD4 population or CD8 population in the total CD3 population in single cells isolated from tumors. Figure 62A shows the percentage of CD4 cells. Figure 62B shows the percentage of CD8 cells. (C-D) shows the differentiation from tumors receiving BCG-disA-OEDot plots with a higher percentage of interferon- γ -producing CD 4T cells in the isolated single cells. FIG. 62C shows INF- γ in CD4 cells ⁺ Percentage of cells. FIG. 62D shows INF- γ in CD8 cells ⁺ Percentage of cells. No significant change in the percentage of interferon-gamma producing CD 8T cells was observed. Briefly, single cells were prepared from excised tumors following 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 (double tail) P<0.05，**P<0.01，***P<0.001，****P<0.0001。

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of many of the terms used in the present invention: singleton et al, dictionary of Microbiology and Molecular Biology (2 nd edition 1994); the Cambridge Dictionary of Science and Technology (Walker, 1988); the Glossary of Genetics, 5 th edition, 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 otherwise specified.

By "agent" is meant any small molecule compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.

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 the methods described herein. As used herein, alteration includes a 10% change in expression level, preferably a 25% change in expression level, more preferably a 40% change, and most preferably a 50% or greater change.

By "improving" is meant reducing, inhibiting, attenuating, reducing, arresting or stabilizing the development or progression of a disease.

By "analog" is meant a molecule that is not identical, but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the function of the analog relative to the naturally-occurring polypeptide. For example, such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog without altering, e.g., ligand binding. In another example, the analog can include an unnatural amino acid.

"cdnP" means 1) a cdnP gene or nucleic acid sequence encoding a cyclic dinucleotide phosphodiesterase (cdnP) protein, or 2) a cyclic dinucleotide phosphodiesterase protein. Examples include the CDnP gene Rv2837c of Mycobacterium tuberculosis (M.tuberculosis) with NCBI gene ID 888920 in H37Rv, and the CDnP protein of UniProtKB/Swiss-Prot P71615.2.

"cGAs" means 1) the cGAs gene or nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein, or 2) the cyclic GMP-AMP synthase protein. Examples of cGAs include homo sapiens (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 human producing 2'3' cGMP. 2'3' cGMP is a STING agonist in humans.

For example, the "cyclase domain" of cGAS refers to a portion or fragment of 522 amino acids of the human cGAS protein described in Kranzusch et al (Cell Reports 2013, 1362-1368PMID 237061). The cyclase domain can be described as having a nucleotidase (NTase) core at amino acids 160-330, and a regulatory-sensor domain, i.e., C-domain, at amino acids 330-522. Mutants of the nucleotidase core sequence, as well as mutants of the regulatory-sensor domain, can be used to produce constitutively active variants of cGAMP that are designed to produce high levels of cGAMP without the general need for activation by DNA binding. Another example of a cyclase domain includes Mycobacterium tuberculosis Rv1354c, NCBI gene ID:887485, and the protein encoded by this gene (UniProtKB/Swiss-Prot: P9WM 13), which encodes a 623 amino acid long protein capable of both c-di-GMP (cyclic di-guanylic acid 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. Mutants of the GGDEF domain and the regulatory-sensor GAF domain and polypeptides truncated to remove the EAL domain (phosphodiesterase activity) may be used to generate constitutively active variants of Rv1354c designed to produce high levels of c-di-GMP.

"DisA" or "DISA" means 1) a DisA gene or nucleic acid sequence encoding a DNA integrity Scan (DisA) protein, or 2) a DNA integrity Scan protein. Examples include the Mycobacterium 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 polyadenylation cyclase of 358 amino acids, such as Dey & Bishai et al Nature Medicine 2015; 21-6. Pmid 25730264. The DisA protein is a diaadenylate cyclase producing c-di-AMP. c-di-AMP is a STING agonist.

By "disease" is meant any condition or disorder that impairs or interferes with the normal function of a cell, tissue or organ. Examples of diseases include, but are not limited to, bladder cancer.

"dncV" means a gene encoding cyclic GMP-AMP synthase that catalyzes the second messenger in cellular signal transduction from GTP and ATP synthesis 3'3' -cyclic GMP-AMP (3'3' -cGAMP). dnCV also produces c-di-AMP and c-di-GMP from ATP and GTP, respectively; however, 3'3' -cGAMP is the major molecule produced in vivo by DncV, compared to 2'3' -cGAMP produced by eukaryotes. dncV is necessary for efficient intestinal colonization by vibrio cholerae (v. Cholerae) and down regulates chemotactic processes that affect colonization. dnCV is inactive against dATP, TTP, UTP and CTP. The DncV protein is a cyclic GMP-AMP synthase from Vibrio cholerae that produces 3' cGAMP. 3'cGAMP 3' is a STING agonist.

By "EAL domain" is meant a conserved protein domain found in a variety of bacterial signaling proteins. The EAL domain may function as a diguanylate phosphodiesterase and has been shown to stimulate degradation of the second messenger cyclic di-GMP. Non-functional EAL domains will not have one or more of these functions. Examples of EAL domains include Mycobacterium tuberculosis Rv1357c, the gene for which is NCBI gene ID:886815, and the 307 amino acid long protein encoded by this gene is UniProtKB/Swiss-Prot: P9WM07, uniProtKB/Swiss-Prot: P9WM07 encodes c-di-GMP Phosphodiesterase (PDE) and comprises a single EAL domain. The activity of this enzyme is to act as a c-di-GMP phosphodiesterase, cleaving a cyclic dinucleotide (which has signaling activity) into 2GMP molecules (which lack signaling activity), as titled "A full-length bifunctional protein involved in c-di-GMP turning over is required for long-term Survival under nutrient constant mutation in Mycobacterium smegmatis", bharati BK, sharma IM, kasett S, kumar M, mukherjee R, chatterji D.Microbiology.2012Jun;158 (Pt 6) described in articles 1415-27.doi. Another example of an EAL domain includes the 336 amino acid long protein in H37Rv encoded by the mycobacterium tuberculosis cdnP gene (Rv 2837 c), a c-di-AMP phosphodiesterase, comprising an EAL domain with the ability to hydrolyze human 2'-3' cgamp (the product of human cGAS enzyme), as described by Jain-Dey Bishai et al Nat Chem biol.2017; 13-217PMID 28106876. The structural features of the EAL domain (cyclic dinucleotide phosphodiesterase activity) and GGDEF domain (cyclic dinucleotide cyclization-biosynthetic activity) are known and well described (e.g., in Schirmer T, jenal U.S. structural and mechanical stabilizers of c-di-GMP signalling. Nat Rev Microbiol.2009; 7.

By "effective amount" is meant the amount required to ameliorate the symptoms of the disease relative to an untreated patient. The effective amount of one or more than one active compound for use in the practice of the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

"dncV" means 1) a dncV gene or nucleic acid sequence encoding a cyclic GMP-AMP synthase (dncV) protein, or 2) a 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.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may comprise, 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.

By "gene deletion" is meant deletion of the entire gene coding region of the gene of interest from the chromosome of BCG using an allelic exchange method well known to those skilled in the art. Gene replacement with a selectable marker, such as an antibiotic resistance cassette, is a form of allelic exchange and can be performed. There are also techniques to generate unlabeled deletions (no selectable marker) in which the gene is completely deleted and no selectable marker is introduced at its location.

By "gene domain deletion" is meant the use of the above allelic exchange method to remove the portion of the gene encoding a particular domain (in the present case, the EAL domain of Rv1354c encoding the CDN phosphodiesterase domain of a multifunctional polypeptide) while leaving the rest of the polypeptide intact and in frame.

"Homo sapiens" means Homo sapiens.

"obtaining" as in "obtaining an agent" means synthesizing, purchasing, or otherwise obtaining the agent.

By "over-expression" is meant, in a general sense, that the number of genes expressing their respective proteins is greater than the wild-type or reference gene. Examples of the gene that produces an overexpressed protein in the present invention include fusion of DNA encoding the gene of interest to a strong promoter such as Phsp60 or a strong conditionally active promoter such as PtetOFF in BCG. In PtetOFF, gene expression is turned off in the presence of tetracycline, anhydrotetracycline, or doxycycline; however, when recombinant BCG is administered as an immunotherapy in humans or animal models, the gene of interest will be turned on. This conditional activity strategy has the following advantages: preventing strong overexpression of the enzymes that produce cyclic dinucleotides during BCG growth may have any detrimental effect on the viability or growth rate of the BCG organism and allow strong expression ("overexpression") only when BCG immunotherapy is administered as a therapeutic agent to a mammalian host.

"Mtb" means Mycobacterium tuberculosis.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides may be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins as well as non-glycoproteins.

"reduce" or "decrease" means, for example, at least about 10%, 25%, 50%, 75%, or 100%, or any percentage negative change therebetween.

By "increase" is meant, for example, a positive change of at least about 10%, 25%, 50%, 75%, or 100%, or any percentage therebetween.

"reference" means a standard or control condition.

"reference sequence" means a defined sequence that is used as a basis for sequence comparison. The reference sequence may be a subset or the entirety of the specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence will typically 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 near or between them.

By "reference BCG strain" is meant, for example, a conventional BCG strain that does not comprise the expression vector of the invention and/or its endogenous genes and is incapable of expressing cdnP functional protein, rv1354c functional protein, rv1357c functional protein, or a combination thereof.

By "ability to recognize regulatory DNA" is meant the ability of a protein to detect or bind to DNA. For example, cGAS proteins are known to bind to DNA, such as cytosolic DNA, and trigger the reaction of GTP and ATP to form cyclic GMP-AMP (cGAMP). cGAMP binds to a stimulator of the interferon gene (STING), which triggers phosphorylation of IRF3 via TBK 1.

"Rv1354c" means 1) an Rv1354c gene or nucleic acid sequence encoding an Rv1354c protein, or 2) an Rv1354c protein (e.g., gupta, kumar, and Chatterji; PLoS ONE (11 months 2010); volume 5; stage 11; and Bhariati, sharma, kasetty, kumar, mukherjee, and Chatterji; microbiology (2012), 158, 1415-1427). The Rv1354c protein is a diguanylate cyclase producing c-di-GMP. c-di-GMP is a STING agonist.

"Rv1357c" means 1) an Rv1357 gene or nucleic acid sequence encoding a cyclic di-GMP phosphodiesterase protein (Rv 1357), or 2) a cyclic di-GMP phosphodiesterase protein (e.g., gupta, kumar, and Chatterji; PLoS ONE (11 months 2010); volume 5; stage 11; and Bhariati, sharma, kasetty, kumar, mukherjee, and Chatterji; microbiology (2012), 158, 1415-1427). The Rv1357c protein is a diguanylate cyclase producing c-di-GMP. c-di-GMP is a STING agonist.

By "STING agonist" is meant a molecule that binds to STING (a stimulator of interferon genes, or TMEM 173), activates STING, and triggers activation of the IRF3-TBK1 pathway, resulting in increased transcription of type 1 interferons and other genes.

"CDN" means a cyclic dinucleotide, such as 3'-5'c-di-AMP, 3'-5'c-di-GMP, 3'-3' cGAMP (also known as 3'-5',3'-5 cGAMP, which is a product of Vibrio cholerae DncV protein), or 2' -3'cGAMP (also known as 2' -5',3' -5 cGAMP, which is a product of human cGAS protein).

"PAMP" means a pathogen-associated molecular pattern (pathogen associated molecular pattern). PAMPs are microbial products, including small molecules that are recognized by innate immune sensors. Examples of PAMP are 3'-5'c-di-AMP, 3'-5'c-di-GMP, 3'-3' cGAMP.

"DAMP" means a risk associated molecular pattern (danger associated molecular pattern). DAMPs are host-derived (i.e., human, mouse, or other mammalian disease model) molecules that are generated to signal a risk, such as infection or other normal physiological disorder. An example of DAMP is 2'-3' cgamp,2'-3' cgamp produced by the host sensor enzyme cGAS upon detection of double stranded DNA in the cytoplasm, as occurs during viral or certain intracellular bacterial infections.

"panCD" means a genetic operon from a bacterium or other species that encodes the biosynthetic gene panC (encoding a PanC protein having pantothenate- β -alanine ligase enzyme activity) and the biosynthetic gene panD (encoding a PanD protein having aspartate 1-decarboxylase enzyme activity). PanC and PanD proteins are involved in the protein which is also referred to as vitamin B ₅ The pantothenic acid or pantothenate salts (a B vitamin) are essential for the biosynthesis. Pantothenic acid, a water-soluble vitamin, is an essential nutrient for bacteria and all mycobacteria, including BCG. Pantothenic acid is required for the synthesis of coenzyme a (CoA) and for the synthesis and metabolism of proteins, carbohydrates and fats.

By "specifically binds" is meant, for example, a compound, nucleic acid, peptide, protein, or antibody that recognizes and binds to a polypeptide or nucleic acid sequence, but does not substantially recognize and bind to other molecules in a sample.

By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequences are at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence identity is commonly determined using sequence analysis software (e.g., genetics Computer Group, university of Wisconsin Biotechnology Center, 17100 sequence analysis software package of Universal Avenue, madison, wis.53705, BLAST, BESTFIT, utah,GAP or PILEUP/PRETTYBOX program). 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; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where e ^-3 And e ^-100 The probability scores in between indicate closely related sequences.

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.

By "sensitivity" is meant the percentage of subjects with a particular disease.

By "specific" is meant the percentage of subjects that are correctly identified as having a particular disease, i.e., normal or healthy subjects. For example, specificity is calculated as the number of subjects with a particular disease compared to normal healthy subjects (e.g., non-cancer subjects).

By "training immunity" is meant the ability of one antigenic stimulant to elicit a stronger immune response against a second, different antigen administered at a later time. Training immunity is antigen-independent, based on heterologous CD4 and CD8 memory activation, cytokine-mediated, and is associated with epigenetic and metabolic changes.

"Phsp60" or "Phsp65" means a strong mycobacterial promoter derived from the Hsp65 UTR of Mycobacterium leprae (Mycobacterium leprae).

"5'UTR" means the 5' untranslated region of a gene.

"3'UTR" means the 3' untranslated region of a gene.

"WT" means a wild type.

"BCG-WT" means a wild-type strain of M.bovis BCG.

The ranges provided herein are to be understood as a shorthand representation of all values falling within the range. For example, a range of 1 to 50 should 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.

As used herein, the terms "treat", "treating" and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be understood that treating a disorder or condition does not require (although does not preclude) that the disorder, condition, or symptoms associated therewith be completely eliminated.

As used herein, the term "or" is understood to be inclusive, unless specified otherwise or clear from the context. The terms "a", "an" and "the" as used herein are to be construed as singular or plural unless expressly stated or apparent from the context.

Unless specifically stated or apparent from the context, the term "about" as used herein is understood to be within the normal tolerance of the art, e.g., within 2 standard deviations of the mean. About may be understood to be 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 the context, all numbers provided herein are modified by the term about.

As used herein, "comprises," "comprising," "contains," "containing," and "having" and similar words may have the meaning ascribed to U.S. patent laws and may mean "including," "including," and similar words; "consisting essentially of … … (consenting essentially of)" or "consisting essentially of" likewise has the meaning ascribed to U.S. patent law, and the terms are open-ended, allowing more presence than stated, as long as the basic or novel features recited are not altered by more presence than stated, but do not include embodiments of the prior art.

Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

As used herein, the terms "preventing", "preventive 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 developing, or is predisposed to developing, the disorder or condition.

Such treatment (surgery and/or chemotherapy) will suitably be administered to a subject, particularly a human, suffering from, susceptible to, or at risk of bladder cancer or a disease, disorder, or symptom thereof. Determining those subjects "at risk" can be done by any objective or subjective determination of the subject's or health care provider's diagnostic tests or opinions (e.g., genetic tests, enzyme or protein markers, markers (as defined herein), family history, etc.). In some embodiments, determining that the subject is predisposed to or has urothelial cancer is determined by measuring the level of at least one marker.

In some embodiments, the invention relates to genetic alterations of mycobacterium bovis BCG (hereinafter, "BCG"), which result in recombinant BCG (hereinafter, "rBCG") strains. These strains have greater potential as (i) tuberculosis vaccines and/or (ii) immunotherapy for non-muscle invasive bladder cancer (NMIBC). Some embodiments of the invention relate to BCG strains that synthesize and secrete high levels of Cyclic Dinucleotides (CDNs), which are known to elicit valuable immunomodulatory responses from human phagocytic cells such as macrophages, dendritic cells, and other cells. Another embodiment of the invention is to combine genetic modifications of BCG to generate multivalent CDN-overexpression modifications, including the addition of new genetic material to synthesize CDN and/or mutations of endogenous BCG phosphodiesterase genes or genetic domains that will enhance the accumulation and release of CDN.

BCG

BCG (BCG vaccine) is a mutant form of M.bovis produced by Calmette and Guerin, french microbiologist, in 1921 by 13 years of serial passage of virulent M.bovis. Between 1921 and 1960, BCG was serially passaged in many laboratories around the world until defined seed lots were established and stored in the reference laboratory. Thus, many variants of BCG exist worldwide, such as BCG Pasteur, BCG Tice, BCG Tokyo, BCG Danish, BCG Montreal, and others. Most existing BCG strains have now been determined by whole genome sequencing. The major differences between virulent mycobacterium bovis and various BCG strains include the deletion of at least 15 distinct regions in BCG that contain genomic deletions compared to virulent mycobacterium tuberculosis. The key differential regions in BCG development are RD1 (9.5 kb deletion, resulting in loss of the Esx-1 secretion system and inability to release the antigens ESAT-6 and CFP-10) and RD3 (9.2 kb deletion). Compared with virulent Mycobacterium tuberculosis, no RD4-RD11 differential region exists in all BCG strains.

BCG has been used as a vaccine for the prevention of Tuberculosis (TB) since the 20 th 20 s of the 20 th century. In 2004, BCG was estimated to be given to about 1 million children, so since its first time put into use, BCG has been given to about 50 million people, and is therefore the most widely used vaccine historically. The most common is intradermal administration at birth and to date it is still administered in most countries except in the united states, canada and parts of europe. BCG has been shown to reduce the incidence of disseminated TB in children, but individuals vaccinated with BCG are not fully protected from the risk of TB.

BCG has also gained widespread use since 1977 as a cancer immunotherapy for non-muscle invasive bladder cancer (NMIBC). BCG is administered intravesically weekly for six weeks, and in some cases such as high risk illness, at 3 months, 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after the initial therapy, BCG is administered weekly as a maintenance therapy for three weeks. Intravesical BCG has been shown to (i) induce infiltration of monocytes comprising primarily CD 4T cells and macrophages, (ii) increase expression of interferon gamma (IFN γ) in the bladder, and (iii) increase levels of urinary cytokines IL-1, IL-2, IL-6, IL-8, IL-12, IFN γ, and TNF α.

Although BCG is widely used globally as (i) a vaccine for TB and (ii) an immunotherapy for NMIBC, there is considerable room for improvement in its efficacy. For TB, BCG provides only partial protection primarily against disseminated tuberculosis in children. For NMIBC, about 30% of patients suffer from BCG-resistant disease. These individuals require higher risk treatment with systemic chemotherapy and have a higher rate of bladder cancer progressing to a more invasive form.

Urothelial cancer

Urothelial cancer of the bladder is the most common malignancy of the urethra. Urothelial cancer is the fourth most common cancer in men and the eleventh most common cancer in women. It is estimated that about 79,000 new cases of bladder cancer will be diagnosed in the us in 2017, associated with 19,870 deaths. Although the estimated five-year survival rate for patients with bladder cancer is 78%, the survival rate is significantly reduced for patients with locally advanced or metastatic disease. Approximately 75% of patients with bladder cancer present with disease restricted to the mucosa (stage Ta, carcinoma in situ) or submucosa (stage T1), 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 later), the first line treatment option is platinum-containing chemotherapy followed by bladder removal. For those patients with NMIBC who do not respond to intravesical treatment, there is a high risk of developing MIBC. Thus, a high recurrence rate and significant risk of progression require the administration of additional therapies. Therefore, improving the clinical outcome of patients with high risk NMIBC requires the development of new treatments.

Intravesical administration of Bacillus Calmette Guerin (BCG) developed for NMIBC in the 70's of the 20 th century provided the first successful immunotherapy against established solid cancers, and it is still the standard of care for patients with NMIBC. (activated EJ, newton MR, O' Donnell MA, luo Y. Black Cancer Immunotherapy: BCG and beyond. Adv Urol.2012; 2012. The exact mechanism of the anti-tumor effect of the attenuated strain BCG of M.bovis is not known, but it is believed that BCG, after binding to the urothelium via fibronectin and integrin α 5 β 1, coordinates strong immune cellular and humoral immune responses, mainly Th1 responses (Redelman-Sidi G, glickman MS, bochner BH. The mechanism of action of BCG therapy for the tablet holder- -a current therapeutic. Nat Rev Urol.2014; 11. However, BCG treatment typically has a complete response rate of 55-65% to papillary tumors and 70-75% to Carcinoma In Situ (CIS). (activated EJ, newton MR, O' Donnell MA, luo Y. Black Cancer Immunotherapy: BCG and beyond. Adv Urel.2012; 2012. Thus, patients with BCG unresponsive and relapsing disease and the burden on treatment-intolerant patients has prompted a need to further improve the efficacy of BCG against NMIBC.

CDNs are important PAMPs and DAMPs that generate valuable immune responses against TB and NMIBC.

Bacterial pathogen-associated molecular patterns (PAMPs).Human cells utilize an innate immune monitoring system called the cytoplasmic monitoring program (CSP) to detect nucleic acids, including cyclic dinucleotides, in the cytoplasm. CSP, originally characterized as a viral defense system, has now been shown to be important in the defense against bacteria, particularly against intracellular bacteria such as mycobacterium tuberculosis, listeria monocytogenes (Listeria monocytogenes), salmonella (Salmonella) species and other bacteria. Cytoplasmic Pattern Recognition Receptors (PRRs), including STING, cGAS, DDX41 and many others, are capable of binding to cytoplasmic 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, STAT6 being independent of the TBK1-IRF3 pathwayInducing chemokines such as CCL2 and CCL20.STING activation is also thought to activate the transcription factor NF κ B through I κ B kinase (IKK) activation.

Human risk associated molecular patterns (DAMPs).Cyclic cGAMP (cGAS) synthase is a cytoplasmic PRR that recognizes cytoplasmic DNA. Cyclic cGAMP (cGAS) synthase undergoes a conformational change upon binding to DNA, which activates its core enzyme activity, catalyzing the formation of 2'3' cGAMP. 2'3' cGAMP followed by an efficient DAMP that activates STING-TBK1-IRF3 axis (axis), resulting in increased expression of type 1 interferon, as well as STAT6 activation and IKK activation.

Mechanism of STING-mediated CDN-triggered immune responses.Type I IFNs produced by both innate immune cells in the tumor microenvironment and by the tumor cells themselves are known to mediate anti-tumor effects against several malignancies because of their ability to intervene in all stages of cancer immune editing. (Zitvogel L, galluzzi L, kepp O, smyth MJ, kroemer G.type I interferons in anticancer importance. Nat Rev Immunol.2015; 15. STING (stimulator of the interferon gene), a major regulator of the type I IFN innate immune response to pathogens after recognition of solute 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 cross roads of immunity, metablism, and disease. Biochim Biophys acta.2015; 1852. 365-78. PMID. Therefore, novel anti-cancer immunotherapy based on recombinant type I IFN, type I IFN-encoding vectors, type I IFN-expressing cells and STING agonists is currently being developed as a novel tumor immunotherapy.

Overexpression of the PAMP immunomodulator 3 '-5'c-di-AMP.3'-5'c-di-AMP is a strong inducer of STING-TBK1-IRF3 axis. 3'-5'c-di-AMP is produced by mycobacteria including BCG through a DISA gene encoding a DisA protein (BCG protein WP _010950916.1, mycobacterium tuberculosis protein Rv3586 or P9WNW5.1 in BCG). Mycobacterium tuberculosis synthesizes and secretes c-di-AMP, which activates Interferon Regulatory Factor (IRF) pathway and type I through STING signal conduction and cGASIFN response. (Ahmed D, cassol E.roll of cellular metabolism in regulating type I interference responses: implications for tumor immunology and treatment. Cancer Lett.2017; 409. C-di-AMP over-expressed Mycobacterium tuberculosis strains show a reduction of TB in a mouse model. As a mucosal adjuvant, c-di-AMP exerts an immunostimulatory effect, leading to maturation of dendritic cells, up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines, and a strong Th1, th17 and CD 8T cell response against pathogens. The BCG strain with c-di-AMP over-expression (rBCG-disA or BCG-disA-OE) has been constructed and surprisingly found to produce significantly higher IRF and IFN- β responses than BCG itself, indicating that bacterially derived c-di-AMP enters the host cell cytoplasm despite the lack of the ESX-1 protein secretion system. (Ahmed D, cassol E.roll of cellular metabolism in regulation type Interferon responses: antibiotics for tumor immunology and treatment. Cancer Lett.2017; 409. These findings indicate that rBCG strains modified to overexpress c-di-AMP can induce better protective immunity against bladder tumors than BCG itself.

Induction of pro-inflammatory Th1 cytokines in mouse bone marrow-derived macrophages (BMDM) in response to BCG overexpressing mycobacterium tuberculosis disA (MT 3692): the Mycobacterium tuberculosis genome encodes the diadenosine cyclase (DisA, also known as DacA, P9WNW5.1 in the UniProtKB/Swiss-Prot database) that synthesizes c-di-AMP from ATP or ADP. The BCG protein WP _010950916.1 (NCBI reference number) is 100% identical to M.tuberculosis DisA. Mycobacterium tuberculosis strains that overexpress DISA poison macrophages by releasing excessive amounts of c-di-AMP, a unique bacterial PAMP that activates STING-dependent IFN- β production. (Ahmed D, cassol E.roll of cellular metabolism in regulation type Interferon responses: antibiotics for tumor immunology and treatment. Cancer Lett.2017; 409. To expand the antigenic repertoire of non-pathogenic vaccine strains (antipathogenic vaccine), BCG Pasteur was transformed with a plasmid conferring kanamycin resistance (Kan-R) carrying the strong mycobacterial promoter P _hsp60 Fused dissa groups from mycobacterium tuberculosisTherefore (M.tuberculosis Rv3586 or MT 3692) (the dis A genes of M.tuberculosis and BCG are 100% identical). Addition of this plasmid to BCG-Pasteur increased the levels of dis A mRNA by 50-fold (FIG. 1 b). The closely related m.tb-disA-OE strain released 15-fold more c-di-AMP into the macrophage cytoplasm compared to wild-type mycobacterium tuberculosis (fig. 1 a), and thus BCG-disA-OE was expected to release significantly more c-di-AMP into the host cytoplasm as well. These DISA overexpressing recombinants (rBCG or BCG-DISA-OE) are better STING-dependent IFN- β inducers than the parental strain. Most importantly, as reported in PCT/US2016/017248, filed on 10/2/2016, the rBCG-inoculated guinea pigs were significantly better protected from aerosol infection with virulent mycobacterium tuberculosis, indicating an improved protective efficacy compared to existing BCG strains.

As shown in FIG. 2, the immune response elicited by BCG-Pasteur disA-OE was tested in an in vitro macrophage infection model. BMDM from C57BL/6 mice infected with BCG-Pasteur disA-OE showed significant upregulation of IFN- β, TNF- α, IL-6, and IL-2 compared to uninfected macrophages or wild type BCG-infected macrophages.

As shown in fig. 3, enhanced c-di-AMP-based STING activation in RAWBlue ISG macrophages was confirmed. RAWBlue macrophages showed increased levels of IRF3 when infected with BCG-Pasteur disA-OE compared to parental controls.

As shown in FIGS. 4A-4C, a significant increase in secreted pro-inflammatory cytokines (TNF-. Alpha., IL-6, and IL-1. Beta.) was found in the culture supernatants of BMDM in BCG-Pasteur-dis A-OE infected mice. These findings indicate that BCG-Pasteur-disA-OE with an increased repertoire of antigens behaves like a STING agonist and is therefore a potent inducer of STING-dependent type I IFN. Furthermore, the immune response in macrophages in response to BCG-Pasteur disA-OE is biased towards Th1, a phenotype largely responsible for the control of NMIBC by BCG immunotherapy.

As shown in FIG. 5, BCG-disA-OE elicited anti-tumor immune responses in human bladder cancer (RT 4) cells. BCG-Pasteur-disA-OE was tested to determine whether BCG-Pasteur-disA-OE elicited a similar immune response in Bladder Cancer (BC) cells as compared to WT strains BCG-Pasteur and Oncotice (the current immunotherapeutic BCG strain). Human RT4 BC cells derived from human NMIBC tumors were challenged with wild-type (both Pasteur and TICE) strains and recombinant BCG Pasteur disA-OE strains with 1. It was found that key immune mediators such as monocyte chemotactic protein 1 (MCP-1)/CCL 2, IFN- β and IL-1 β were significantly increased in bladder cancer cells exposed to BCG-Pasteur-dis A-OE compared to the response to the wild type strain.

As shown in fig. 6, an experimental system was established to test whether intravesical BCG-dis-a-OE immunotherapy resulted in an increased Th1 response and anti-tumor efficacy in the MNU oncogenic model of NMIBC. The results from the above mentioned experiments using RT4 cells encouraged the present inventors to test the relative therapeutic efficacy of BCG-Pasteur disA OE in the rat NMIBC model in vivo, which was pioneered in the Bivalacqua laboratory. (Kates M, nirschl T, sopko NA, matsui H, kochel CM, reis LO, netto GJ, hoque MO, hahn NM, mcConkey DJ, baras AS, drake CG, bivalacqua TJ. Intragenic BCG indexes CD4 (+) T-Cell Expansion in an Immune company Model of blower cancer Immunol Res.2017; 5. In this model, an oncogenic alkylating agent, N-methyl-N-nitrosourea (MNU), was used to induce urothelial cancer in female Fischer rats.

As can be observed in fig. 7, BCG-disA-OE had significant immunotherapeutic effects in the rat bladder cancer model. Urothelial dysplasia develops within 8 weeks of MNU instillation, and by 16 weeks after the first instillation, all rats show carcinoma in situ, papillary Ta or high grade T1 urothelial carcinoma with histopathological and immunophenotypic characteristics similar to those observed in human urothelial carcinoma. Using this model, intravesical BCG immunotherapy was shown to result in CD4 in the urothelium ⁺ A large transient increase in the population of T cells. (Kates M, nirschl T, sopko NA, matsui H, kochel CM, reis LO, netto GJ, hoque MO, hahn NM, mcConkey DJ, baras AS, drake CG, bivalacqua TJ. Intra scientific BCG Induces CD4 (+) T-Cell Expansion in an Immune company Model o(f Bladder cancer. Cancer Immunol Res.2017; 5-594. PMID. Intravesical instillation of BCG-disA-OE strain was performed in MNU-treated rats starting 8 weeks after MNU induction when tumors were visible, and was administered continuously weekly for 6 weeks. Bladder tumors were staged by GU pathologists according to WHO-ISUP classification, and percent tumor involvement (sum of Ta, T1 and CIS) was calculated for each group according to criteria as described. (Kates M, nirschl T, sopko NA, matsui H, kochel CM, reis LO, netto GJ, hoque MO, hahn NM, mcConkey DJ, baras AS, drake CG, bivalacqua TJ. Intragenic BCG indexes CD4 (+) T-Cell Expansion in an Immune company Model of blower cancer Immunol Res.2017; 5. It was found that the tumor involvement index in rats treated with BCG-Pasteur disA-OE was significantly reduced compared to the bladder from untreated rats or BCG-Pasteur treated rats.

As can be observed in FIG. 8, BCG-disA-OE induced characteristic cytokine and chemokine signatures in rat bladders undergoing immunotherapy (pathological cytokine and chemokine signature). The urinary bladder of rats from rats treated with BCG-disA-OE showed significant induction of IFN- α/β, IFN- γ, IL-1 β, TNF- α, TGF- β, iNOS, IP-10, MCP-1 and MIP-1 α compared to untreated rats or BCG-Pasteur treated rats.

As shown in FIG. 9, CCL2 was found in the bladder of rats treated with BCG-Pasteur-DISA-OE ⁺ Macrophages, nos2 ⁺ And IL-1. Beta ⁺ Evidence of increased infiltration of M1 macrophages, which is accompanied by increased IL-6 and IFN-expression. Interestingly, increased levels of IP-10 were found, which together with increased IFN- γ was known to promote strong T cell recruitment at the site of infection and inflammation.

FIG. 10 shows a summary of the changes observed in cytokine expression levels of BCG-disA-OE versus BCG-WT in primary cells, cancer cell lines, and in rat bladder cancer tissue. As can be observed, BCG-disA-OE significantly upregulated cytokines associated with Th 1T cell and M1 macrophage expansion, two type 1 interferons and three pro-inflammatory chemokines (2-fold to 30-fold) in these cells, cell lines and tissues compared to BCG-WT. In contrast, BCG-disA-OE generally down-regulates cytokines associated with Th 2T cell and M2 macrophage expansion (1-fold to 10-fold) compared to BCG-WT.

BCG immunotherapy may be effective via three immune mechanisms: (ii) a cytokine milieu that promotes macrophage-mediated activation of CD4 cells against a tumor antigen, and (iii) macrophage M1 conversion that promotes enhanced tumoricidal activity. The findings reported herein strongly suggest that BCG overexpressing c-di-AMP is taken up by bladder tumor cells and myeloid cells, which reside in or are recruited to the tumor microenvironment, and that BCG induces activation of host immune responses, including STING and type I IFN responses, as well as NF- κ B signaling that promotes secretion of cytokines and chemokines, macrophage recruitment, and apoptotic mechanisms, all of which collectively reduce tumor progression.

In addition to overexpression of dispa resulting in increased levels of the PAMP molecule c-di-AMP, there are additional recombinant DNA modifications that can be made to BCG to enhance the production of other PAMPs and DAMP molecules. Genes for other CDN cyclases- - (i) the GGDEF domains of BCG _ RS07340 protein or Mycobacterium tuberculosis Rv1354c protein (100% identical to each other), - - (ii) the Vibrio cholerae DncV protein, which is Q9KVG in Swiss-Prot, which is 2'-5'c-GAMP synthase, and (iii) the human cGAS protein Q8N884, which is 2'-3' cGAMP synthase- -can be added to BCG. These CDN cyclase genes added may be added individually or in combination. Such a combination would represent a multivalent CDN over-expressing BCG. In addition, as shown in fig. 11, BCG has several CDN phosphodiesterase genes or genes containing phosphodiesterase domains. These endogenous phosphodiesterase genes and intragenic phosphodiesterase domains are removed using recombinant technology methods: (i) the BCG WP _003414507 gene which encodes a CDN PDE in BCG that is 100% identical to mycobacterium tuberculosis Rv2837c (also known as CdnP or CnpB), (ii) DNA encoding the EAL domain of the protein BCG RS07340 (previous BCG _1416 c) which is 100% identical to the known CDN PDE mycobacterium tuberculosis Rv1354c protein, and (iii) the gene encoding BCG AHM07112 which is homologous to the known CDN PDE mycobacterium tuberculosis Rv1357 c. Removal of the genes encoding these PDEs would serve to further increase the levels of CDN PAMP and DAMP molecules produced by the rBCG strains disclosed herein.

SEQ ID NO:1

The amino acid sequence of the diadenosine cyclase DISA from BCG and other related mycobacteria (358 amino acids; BCG protein AOQ _ RS18745; NCBI reference sequence: NZ _ CUWL 01000001.1). The same sequence is present in other BCG strains, for example in mycobacterium tuberculosis such as protein Rv3586 or MT3692, and in mycobacterium bovis such as protein Mb3617.MHAVTRPTLREAVARLAPGTGLRDGLERILRGRTGALIVLGHDENVEAICDGGFSLDVRYAATRLRELCKMDGAVVLSTDGSRIVRANVQLVPDPSIPTDESGTRHRSAERAAIQTGYPVISVSHSMNIVTVYVRGERHVLTDSATILSRANQAIATLERYKTRLDEVSRQLSRAEIEDFVTLRDVMTVVQRLELVRRIGLVIDYDVVELGTDGRQLRLQLDELLGGNDTARELIVRDYHANPEPPSTGQINATLDELDALSDGDLLDFTALAKVFGYPTTTEAQDSTLSPRGYRAMAGIPRLQFAHADLLVRAFGTLQGLLAASAGDLQSVDGIGAMWARHVREGLSQLAESTISDQ

SEQ ID NO:2

The DNA sequence of the diadenosine cyclase disA from BCG and other related mycobacteria (1077 nucleotides [358 codons, 1 stop codon ]; encoding BCG gene AOQ _ RS18745; NCBI reference sequence: NZ _ CUWL 01000001.1). The same sequence is present in other BCG strains, for example in Mycobacterium tuberculosis such as the gene Rv3586 or MT3692, and in Mycobacterium bovis such as the gene Mb3617.

SEQ ID NO:3。

Plasmid pSD5B-P _hsp60 DisA is an episomally replicating Escherichia coli (E.coli) -Mycobacterium shuttle plasmid, derived from P _hsp60 The promoter overexpresses the BCG disA gene, the DNA sequence (7742 nucleotides; promoter P which will contain a portion of nucleotides 13 to 180 of the mycobacterium leprae hsp65 gene _hsp60 DNA is underlined; nucleotides 242 to 1318 of a dis coding sequence; the ATG start codon and the TAA stop codon are shown in bold, underlined)。

Mycobacteria that overexpress disA have attenuated virulence. As shown in FIG. 12, 3.5log was used when passing through the aerosol route ₁₀ Unit of vector pSD5B P _hsp60 When mice are infected with Mycobacterium tuberculosis of dis A plasmids (M.tb-dis A-OE or Mtb-OE) or wild type Mycobacterium tuberculosis (Mtb-CDC 1551), the median time to death (MTD) of the animals is obviously different. As can be observed, the wild type Mycobacterium tuberculosis (Mtb-CDC 1551) gave an MTD of 150.5 days, while carrying pSD5BP _hsp60 Mycobacterium tuberculosis of the disA plasmid (M.tb-disA-OE or Mtb-OE) is a significantly weaker pathogen, giving an MTD of 321.5 days. A similar reduction in the pathogenicity of BCG-disA-OE is expected compared to BCG-WT. Thus, it is likely that if BCG-disA-OE is used as a cancer immunotherapy, one would expect a reduced rate of blood flow dissemination, reduced dysuria, reduced urgency and reduced fatigue compared to BCG-WT.

The PAMP immunomodulator 3'-5'c-di-GMP has been produced by overexpressing the GGDEF domain of the protein BCG _ RS07340 Expression of.3'-5'c-di-GMP is a strong inducer of STING-TBK1-IRF3 axis. 3'-5'c-di-GMP is produced by mycobacteria (including BCG) via the GGDEF domain of the protein BCG _ RS07340 (formerly BCG _1416 c) and via the Mycobacterium tuberculosis Rv1354c gene. The BCG _ RS07340 protein (100% identical to Mycobacterium tuberculosis Rv1354c protein) encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase. Thus, part of functioning as diguanylate cyclase is the endogenous CDN-producing enzyme in BCG. The length of the full-length BCG _ RS07340 polypeptide is 623Amino acids, and the domain structure thereof is: N-terminal-GAF-GGDEF-EAL-C-terminal. The GAF domain (about amino acids 1-190) is a regulatory domain that affects the activity of other domains. The GGDEF domain (about amino acids 190-350) is a diguanylate cyclase that catalyzes the reaction 2GTP → c-di-GMP +2 pyrophosphate. The EAL domain (about amino acids 350-623) is a diguanylate phosphodiesterase that catalyzes the reaction c-di-GMP → 2 GMP. By genetically removing the DNA sequence encoding the C-terminal EAL domain, DNA encoding the GGDEF domain can be used to generate recombinant BCG that will overexpress diguanylate cyclase activity. This can be accomplished by also deleting the DNA encoding the regulator-sensor GAF domain and/or using mutations in the DNA encoding the GAF domain to mitigate any cyclase inhibitory activity it may have. Such a technique to produce a constitutively active recombinant form of the BCG _ RS07340 protein would produce high levels of c-di-GMP in recombinant BCG.

SEQ ID NO:4

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 WP003898837.1; old locus tag BCG _1416 c). The same sequence is present in other BCG strains, for example in Mycobacterium tuberculosis such as the protein Rv1354c or MT1397, and in Mycobacterium bovis such as the protein Mb1389c. The EAL domain is amino acids 354 to 623 and is underlined.

SEQ ID NO:5

Bifunctional diguanylate cyclase/phosphodiesterase BCG _ RS07340, DNA sequence (1872 nucleotides [623 codons +1 stop codons ]; encoding BCG protein BCG _ RS07340; NCBI reference sequence: NC _008769.1; protein ID WP003898837.1; old locus tag BCG _1416c; DNA from NC _008769.1 cc 1548390-1546519 mycobacterium bovis BCG Pasteur 1173P 2) from BCG and other related mycobacteria. The same sequence is present in other BCG strains, for example in Mycobacterium tuberculosis such as the protein Rv1354c or MT1397, and in Mycobacterium bovis such as the protein Mb1389c. The EAL domain is encoded by nucleotides 1060 to 1872 and is underlined.

ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGTAGCCAACCAGCTCATGACAG

TCGACGCTGCCACGTCAGCCGAAGTCAGTCAGCGCGTTTTGGCCTATCTAGTGGAACAGCTGGGCGTAGA

TGTCAGCTTTTTGCGTCATAACGATCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATGGCCACCTCGC

CTCAACATACCGGACCCCGATCCGCTCAGGCTGATCTACTTCGCTGATGCCGACCCGGTGTTTGCGCTAT

GCGAACACGCCAAAGAGCCTCTCGTGTTCCGGCCCGAGCCGGCCACCGAGGACTATCAACGCCTCATCGA

AGAAGCCCGCGGGGTTCCGGTAACGTCGGCTGCCGCCGTGCCGCTGGTATCTGGCGAGATCACCACTGGA

CTGCTGGGGTTCATCAAGTTCGGTGATCGGAAATGGCACGAGGCCGAGCTTAACGCCCTCATGACCATCG

CTACACTCTTCGCCCAGGTGCAGGCTCGCGTCGCCGCCGAGGCGCGGCTTCGCTATCTGGCCGACCATGA

CGATCTGACCGGACTGCATAACCGTCGCGCGTTGCTGCAGCACCTGGACCAAAGACTGGCCCCCGGACAA

CCTGGCCCGGTCGCGGCGCTATTTCTCGACTTGGACCGCCTCAAGGCCATCAACGACTACCTGGGCCACG

CCGCCGGTGACCAGTTCATCCATGTGTTCGCCCAACGGATCGGTGACGCACTCGTTGGCGAGAGCCTGAT

CGCCCGACTCGGCGGCGACGAATTCGTCCTCATACCCGCATCTCCAATGAGTGCCGATGCCGCTCAACCG

CTCGCCGAACGTCTTCGCGACCAGCTCAAGGACCACGTCGCTATCGGCGGTGAGGTGCTCACCCGCACCG

TCAGTATCGGTGTCGCCTCAGGGACTCCCGGACAGCACACACCGTCGGACCTCCTGCGCCGAGCCGACCA

AGCCGCTCTGGCAGCCAAACACGCCGGCGGAGATAGCGTCGCGATTTTCACCGCGGACATGTCGGTCAGC

GGCGAACTGCGCAACGATATTGAACTACACCTTCGACGTGGTATCGAATCCGACGCCCTTCGCCTGGTC T

ACCTACCCGAGGTCGACCTACGGACCGGCGACATTGTCGGGACCGAGGCATTGGTCCGGTGGCAGCACC C

CACCCGTGGGCTGCTGGCACCGGGCTGCTTCATCCCTGTGGCCGAATCCATCAACCTTGCAGGCGAATT G

GATAGATGGGTGCTGCGGAGGGCCTGCAATGAATTCTCCGAGTGGCAGTCAGCCGGTTTGGGCCACGAC G

CGCTGCTGCGTATCAACGTCTCAGCTGGACAGCTGGTGACGGGCGGGTTTGTTGACTTCGTCGCAGACA C

GATCGGCCAGCACGGTCTGGACGCCTCGTCCGTGTGTTTGGAAATCACCGAAAACGTTGTGGTGCAAGA C

CTACATACCGCCAGAGCCACCCTGGCTCGACTCAAAGAAGTCGGCGTTCACATCGCTATCGACGATTTC G

GCACCGGCTATAGCGCCATATCACTGTTGCAGACGCTACCGATCGACACGCTCAAGATCGACAAAACAT T

CGTGCGGCAACTCGGAACCAACACTAGCGATCTGGTCATTGTGCGCGGCATCATGACACTCGCCGAAGG C

TTCCAACTCGATGTAGTAGCCGAAGGCGTCGAGACCGAGGCTGCCGCCAGAATTCTATTGGATCAGCGC T

GTTACCGTGCGCAAGGCTTCTTGTTCTCCCGGCCTGTCCCCGGGGAGGCCATGCGGCACATGTTGTCCG C

ACGACGACTACCGCCGACCTGCATACCTGCAACTGACCCGGCGTTATCTTGA

SEQ ID NO:6

Modified bifunctional diguanylate cyclase/phosphodiesterases from BCG and other related mycobacteria, lacking the EAL domain, such that they act as monofunctional diguanylate cyclases, amino acid sequence (353 amino acids; a fragment of BCG protein BCG _ RS07340; NCBI reference sequence: NC _008769.1; protein ID WP003898837.1; old locus tag BCG _1416 c). The same sequence fragments are present in other BCG strains, for example in Mycobacterium tuberculosis such as the protein Rv1354c or MT1397, and in Mycobacterium bovis such as the protein Mb1389c.

SEQ ID NO:7

Modified bifunctional diguanylate cyclase/phosphodiesterases from BCG and other related Mycobacteria, wherein the sequence encoding its EAL domain is deleted such that it encodes a monofunctional diguanylate cyclase, a DNA sequence (1059 nucleotides [353 codons +0 stop codons ]; a fragment encoding the BCG protein BCG _ RS07340; NCBI reference sequence NC-008769.1; protein ID WP003898837.1; old locus tag BCG-1416 c; DNA from NC-008769.1, c1548390-1546519 Mycobacterium bovis BCG Pasteur 1173P 2). The same sequence is present in other BCG strains, for example in fragments of the genes Rv1354c or MT1397 in M.tuberculosis and of the gene Mb1389c in M.bovis.

ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGTAGCCAACCAGCTCATGACAG

TCGACGCTGCCACGTCAGCCGAAGTCAGTCAGCGCGTTTTGGCCTATCTAGTGGAACAGCTGGGCGTAGA

TGTCAGCTTTTTGCGTCATAACGATCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATGGCCACCTCGC

CTCAACATACCGGACCCCGATCCGCTCAGGCTGATCTACTTCGCTGATGCCGACCCGGTGTTTGCGCTAT

GCGAACACGCCAAAGAGCCTCTCGTGTTCCGGCCCGAGCCGGCCACCGAGGACTATCAACGCCTCATCGA

AGAAGCCCGCGGGGTTCCGGTAACGTCGGCTGCCGCCGTGCCGCTGGTATCTGGCGAGATCACCACTGGA

CTGCTGGGGTTCATCAAGTTCGGTGATCGGAAATGGCACGAGGCCGAGCTTAACGCCCTCATGACCATCG

CTACACTCTTCGCCCAGGTGCAGGCTCGCGTCGCCGCCGAGGCGCGGCTTCGCTATCTGGCCGACCATGA

CGATCTGACCGGACTGCATAACCGTCGCGCGTTGCTGCAGCACCTGGACCAAAGACTGGCCCCCGGACAA

CCTGGCCCGGTCGCGGCGCTATTTCTCGACTTGGACCGCCTCAAGGCCATCAACGACTACCTGGGCCACG

CCGCCGGTGACCAGTTCATCCATGTGTTCGCCCAACGGATCGGTGACGCACTCGTTGGCGAGAGCCTGAT

CGCCCGACTCGGCGGCGACGAATTCGTCCTCATACCCGCATCTCCAATGAGTGCCGATGCCGCTCAACCG

CTCGCCGAACGTCTTCGCGACCAGCTCAAGGACCACGTCGCTATCGGCGGTGAGGTGCTCACCCGCACCG

TCAGTATCGGTGTCGCCTCAGGGACTCCCGGACAGCACACACCGTCGGACCTCCTGCGCCGAGCCGACCA

AGCCGCTCTGGCAGCCAAACACGCCGGCGGAGATAGCGTCGCGATTTTCACCGCGGACATGTCGGTCAGC

GGCGAACTG

Overexpression of the PAMP immunomodulator 2'-5'c-GAMP synthase: q9KVG (Swiss-Prot).2'-5'c-GAMP is a strong inducer of STING-TBK1-IRF3 axis. The Vibrio cholerae Q9KVG protein (436 amino acids) encoded by the dncV gene is a known 2'-5'c-GAMP synthase. Recombinant dncV genes optimized for BCG codons can be generated. Codon-optimized structural genes can be overexpressed in BCG by fusion to strong promoters (such as Phsp 60) or conditionally active strong promoters such as PTET-off. Such a technique to produce a constitutively active recombinant form of the Q9KVG protein would produce high levels of 2'-5'c-GAMP in recombinant BCG.

SEQ ID NO:8

The cyclic GMP-AMP synthase, dncV, from Vibrio cholerae, amino acid sequence (436 amino acids; uniProtKB/Swiss-Prot protein ID Q9KVG7.1).

MRMTWNFHQYYTNRNDGLMGKLVLTDEEKNNLKALRKIIRLRTRDVFEEAKGIAKAVKKSALTFEIIQEK

VSTTQIKHLSDSEQREVAKLIYEMDDDARDEFLGLTPRFWTQGSFQYDTLNRPFQPGQEMDIDDGTYMPM

PIFESEPKIGHSLLILLVDASLKSLVAENHGWKFEAKQTCGRIKIEAEKTHIDVPMYAIPKDEFQKKQIA

LEANRSFVKGAIFESYVADSITDDSETYELDSENVNLALREGDRKWINSDPKIVEDWFNDSCIRIGKHLR

KVCRFMKAWRDAQWDVGGPSSISLMAATVNILDSVAHDASDLGETMKIIAKHLPSEFARGVESPDSTDEK

PLFPPSYKHGPREMDIMSKLERLPEILSSAESADSKSEALKKINMAFGNRVTNSELIVLAKALPAFAQEP

SSASKPEKISSTMVSG

SEQ ID NO:9

Circular GMP-AMP synthase DncV from Vibrio cholerae, DNA sequence (1311 nucleotides [436 codons +1 stop codon ]; encoding UniProtKB/Swiss-Prot protein ID Q9KVG7.1; NCBI reference sequence: NC-002505.1: vibrio cholerae O1 biovar Eltor str.N16961 chromosome I, complete sequence, and nucleotides 180419-181729)

GTGAGAATGACTTGGAACTTTCACCAGTACTACACAAACCGAAATGATGGCTTGATGGGCAAGCTAGTTC

TTACAGACGAGGAGAAGAACAATCTAAAGGCATTGCGTAAGATCATCCGCTTAAGAACACGAGATGTATT

TGAAGAAGCTAAGGGTATTGCCAAGGCTGTGAAAAAAAGTGCTCTTACGTTTGAAATTATTCAGGAAAAG

GTGTCAACGACCCAAATTAAGCACCTTTCTGACAGCGAACAACGAGAAGTGGCTAAGCTTATTTACGAGA

TGGATGATGATGCTCGTGATGAGTTTTTGGGATTGACACCTCGCTTTTGGACTCAGGGAAGCTTTCAGTA

TGACACGCTGAATCGCCCGTTTCAGCCTGGTCAAGAAATGGATATTGATGATGGAACCTATATGCCAATG

CCTATTTTTGAGTCAGAGCCTAAGATTGGTCATTCTTTACTAATTCTTCTTGTTGACGCGTCACTTAAGT

CACTTGTAGCTGAAAATCATGGCTGGAAATTTGAAGCTAAGCAGACTTGTGGGAGGATTAAGATTGAGGC

AGAGAAAACACATATTGATGTACCAATGTATGCAATCCCTAAAGATGAGTTCCAGAAAAAGCAAATAGCT

TTAGAAGCAAATAGATCATTTGTTAAAGGTGCCATTTTTGAATCATATGTTGCAGATTCAATTACTGACG

ATAGTGAAACTTATGAATTAGATTCAGAAAACGTAAACCTTGCTCTTCGTGAAGGTGATCGGAAGTGGAT

CAATAGCGACCCCAAAATAGTTGAAGATTGGTTCAACGATAGTTGTATACGTATTGGTAAACATCTTCGT

AAGGTTTGTCGCTTTATGAAAGCGTGGAGAGATGCGCAGTGGGATGTTGGAGGTCCGTCATCGATTAGTC

TTATGGCTGCAACGGTAAATATTCTTGATAGCGTTGCTCATGATGCTAGTGATCTCGGAGAAACAATGAA

GATAATTGCTAAGCATTTACCTAGTGAGTTTGCTAGGGGAGTAGAGAGCCCTGACAGTACCGATGAAAAG

CCACTCTTCCCACCCTCTTATAAGCATGGCCCTCGGGAGATGGACATTATGAGCAAACTAGAGCGTTTGC

CAGAGATTCTGTCATCTGCTGAGTCAGCTGACTCTAAGTCAGAGGCCTTGAAAAAGATTAATATGGCGTT

TGGGAATCGTGTTACTAATAGCGAGCTTATTGTTTTGGCAAAGGCTTTACCGGCTTTCGCTCAAGAACCT

AGTTCAGCCTCGAAACCTGAAAAAATCAGCAGCACAATGGTAAGTGGCTGA

Overexpression of DAMP immunomodulator 2'-3' cGAMP synthase: Q8N884 (Swiss-Prot).2'-3' cGAMP is a strong inducer of STING-TBK1-IRF3 axis. The cGAS protein is produced by the human cGAS gene to produce a 522 amino acid polypeptide that senses cytosolic DNA and functions as a 2'-3' cgamp synthase. When cGAS binds to DNA, the synthase or cyclase domain of cGAS becomes activated. It is possible to produce recombinant cGAS genes which contain only the cyclase domain and are therefore constitutively active. The recombinant gene can also be codon optimized for BCG. Codon-optimized structural genes can be overexpressed in BCG by fusion to strong promoters (such as Phsp 60) or conditionally active strong promoters such as PTET-off. Such a technique to produce constitutively active recombinant forms of cGAS protein would produce high levels of 2'-3'c-GAMP in recombinant BCG.

SEQ ID NO:10

Circular 2'3' -GMP-AMP synthase cGAS, amino acid sequence from homo sapiens (522 amino acids, uniProtKB/Swiss-Prot protein ID Q8N884.2).

MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE

RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD

VPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLN

TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF

RKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK

QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL

KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL

FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF

SEQ ID NO:11

Circular 2'3' -GMP-AMP synthase cGAS from homo sapiens, DNA sequence of mRNA with nucleotide T replacing U (1802 nucleotides; encoding UniProtKB/Swiss-Prot protein ID Q8N884.2; NCBI reference sequence: NM-138441.2. Encoding sequence 1569 nucleotides [522 codons, 1 stop codon ], start codon ATG [ bold, underlined ] at nucleotide 140; stop codon TGA (bold, underlined) at nucleotide 1706).

SEQ ID NO:12

Circular 2'3' -GMP-AMP synthase, cGAS, from homo sapiens, with mycobacterial codon optimization, DNA sequence. (1569 nucleotides [522 codons, 1 stop codon ]; encodes the UniProtKB/Swiss-Prot protein ID Q8N884.2).

ATGCAACCATGGCACGGGAAAGCCATGCAGCGTGCGAGCGAAGCCGGGGCGACGGCCCCCAAGGCGTCGGCGCGTAACGCGCG

GGGTGCGCCCATGGACCCGACGGAGTCCCCCGCGGCGCCGGAGGCGGCCCTGCCGAAAGCGGGTAAGTTCGGTCCAGCGCGGA

AAAGCGGGAGCCGCCAAAAGAAGTCCGCGCCCGACACCCAGGAGCGTCCCCCGGTCCGGGCCACCGGCGCGCGTGCCAAAAAA

GCCCCGCAACGGGCGCAAGATACGCAGCCAAGCGATGCGACCTCCGCCCCCGGGGCGGAGGGTCTGGAGCCCCCGGCCGCCCG

GGAGCCAGCGCTCTCGCGCGCGGGTTCCTGCCGTCAGCGGGGCGCGCGGTGTTCCACGAAACCCCGTCCCCCACCAGGTCCCT

GGGACGTGCCGTCGCCGGGTTTGCCGGTGAGCGCGCCAATCCTGGTCCGGCGCGACGCGGCCCCGGGGGCGTCGAAATTGCGT

GCGGTGCTCGAGAAATTGAAGTTGTCGCGCGACGACATCTCCACGGCCGCGGGTATGGTCAAGGGCGTGGTCGATCATTTGTT

GTTGCGGCTCAAGTGTGATTCGGCGTTCCGCGGGGTGGGCTTGCTGAACACGGGGTCCTACTATGAGCATGTCAAAATCAGCG

CCCCCAACGAATTTGACGTGATGTTTAAGCTGGAAGTGCCACGTATCCAATTGGAAGAGTATTCCAATACCCGTGCGTATTAT

TTCGTCAAATTTAAGCGCAATCCGAAGGAAAATCCACTCAGCCAATTCTTGGAGGGCGAAATTCTGTCGGCCTCGAAAATGCT

CTCCAAATTTCGTAAGATTATCAAGGAGGAGATCAACGACATTAAGGACACGGATGTGATCATGAAACGTAAACGTGGCGGTT

CCCCCGCGGTGACGCTCCTCATTTCGGAAAAAATTTCGGTGGACATTACCCTGGCGTTGGAATCGAAGTCCAGCTGGCCGGCG

TCGACCCAGGAGGGCCTGCGGATTCAAAACTGGTTGAGCGCCAAAGTGCGGAAGCAGCTGCGTCTCAAACCCTTTTATTTGGTC

CCGAAACATGCCAAAGAGGGTAACGGTTTTCAAGAGGAAACCTGGCGTTTGAGCTTCTCCCACATTGAGAAGGAGATTTTGAAC

AACCATGGTAAGTCCAAAACGTGCTGCGAGAATAAGGAAGAAAAATGTTGTCGCAAAGATTGTCTCAAATTGATGAAATATTTG

CTGGAACAACTCAAAGAGCGTTTTAAGGACAAGAAGCATCTCGACAAGTTCTCCTCGTATCACGTCAAGACCGCCTTCTTTCAT

GTCTGTACGCAGAACCCGCAAGATAGCCAGTGGGATCGCAAGGACTTGGGGTTGTGTTTTGACAATTGCGTCACCTATTTCTTG

CAATGTTTGCGGACCGAGAAATTGGAGAACTACTTTATTCCAGAATTCAACTTGTTTTCCTCGAATCTGATTGACAAACGCTCCAAAGAGTTTCTGACGAAGCAGATTGAATACGAGCGTAACAATGAGTTTCCGGTCTTTGACGAGTTTTGA

SEQ ID NO:13

Plasmid pMH94H-P _hsp60 hcGASCo mCherry is a shuttle plasmid of Escherichia coli-mycobacterium _hsp60 The promoter overexpresses the BCG disA gene, the human cGAS gene (with mycobacterial codon optimization) and mCherry, DNA sequences. When introduced into BCG, mycobacterium tuberculosis, mycobacterium bovis or highly related strains, the plasmid integrates into the Mycobacterium chromosome in a single copy (10842 nucleotides; promoter P comprising a portion of nucleotides 901 to 1068 of the hsp65 gene of Mycobacterium leprae _hsp60 The DNA is underlined; the sequence of the disA is nucleotides 1069-2145; human cGAS with mycobacterial codon-optimized sequence is nucleotides 2158 to 3726; the ATG start codon and the TAA or TGA stop codon are shown in bold, underlined).

Knock-out of endogenous BCG phosphodiesterase gene and intragenic segments encoding phosphodiesterase domains to augment CDN PAMP and DAMP levels

Overexpression of CDN by knock-out of endogenous BCG phosphodiesterase WP _003414507

The BCG AHM08589.1 protein encodes the endogenous 316 amino acid bifunctional C-di-AMP and cGAMP phosphodiesterase in BCG 100% identical to the 316 amino acids on the C-terminus of mycobacterium tuberculosis Rv2837C (also known as CdnP, cnpB, 3 '-to-5' oligoribonuclease a, bifunctional oligoribonuclease or PAP phosphatase NrnA). Mycobacterium tuberculosis Rv2837c is known to proteolyze both 3'-5'c-di-AMP (bacterial PAMP molecule) and 2'-3' cGAMP (host DAMP molecule). Since the 315 amino acids of the BCG protein are 100% identical at the C-terminus, knockout (gene replacement) of the BCG AHM08589.1 protein will result in increased CDN (3 '-5'c-di-AMP and 2'-3' cgamp) levels in the recombinant BCG.

SEQ ID NO:14

Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP from BCG (also known as CnpB, 3 '-to-5' oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA), amino acid sequence (316 amino acids; BCG protein AHM08589.1; NCBI reference DNA sequence: CP003494.1, from BCG strain ATCC 35743. Similar sequences are present in Mycobacterium tuberculosis such as protein Rv2837c or MT2903, and M.bovis such as protein Mb2862c.

MDAVGAAALLSAAARVGVVCHVHPDADTIGAGLALALVLDGCGKRVEVSFAAPATLPESLRSLPG

CHLLVRPEVMRRDVDLVVTVDIPSVDRLGALGDLTDSGRELLVIDHHASNDLFGTANFIDPSADSTT

TMVAEILDAWGKPIDPRVAHCIYAGLATDTGSFRWASVRGYRLAARLVEIGVDNATVSRTLMDSHP

FTWLPLLSRVLGSAQLVSEAVGGRGLVYVVVDNREWVAARSEEVESIVDIVRTTQQAEVAAVFKEV

EPHRWSVSMRAKTVNLAAVASGFGGGGHRLAAGYTTTGSIDDAVASLRAALG

SEQ ID NO:15

Bifunctional c-di-AMP and cGAMP phosphodiesterase genes from BCG, cdnP (also known as cnpB or the gene of 3 '-to-5' oligoribonuclease A, bifunctional oligoribonuclease or PAP phosphatase NrnA), DNA sequences (951 nucleotides [316 codons and 1 stop codon ]; encoding the BCG protein AHM08589.1; NCBI reference sequence: CP003494.1, from BCG strain ATCC 35743). Similar sequences are present in Mycobacterium tuberculosis, encoding the proteins Rv2837c or MT2903, and in Mycobacterium bovis, encoding the protein Mb2862c.

GTGGACGCCGTCGGTGCCGCTGCGCTGTTGTCGGCCGCTGCCAGGGTCGGGGTAGTCTGCCACG

TCCACCCCGATGCCGACACCATCGGCGCCGGATTGGCATTGGCATTGGTGTTGGACGGGTGCGG

CAAGCGGGTAGAGGTCAGCTTTGCCGCGCCGGCGACACTGCCCGAGTCGCTGCGTTCGCTGCCG

GGCTGCCATCTGCTGGTCCGCCCTGAGGTGATGCGCCGCGATGTCGATTTGGTTGTGACTGTTGA

CATTCCGAGTGTTGATCGGCTCGGTGCTCTGGGCGATCTAACTGATTCCGGGCGGGAGCTCCTG

GTAATCGACCATCACGCCTCCAACGACCTGTTCGGCACCGCGAATTTCATTGACCCGTCGGCGG

ATTCCACCACGACGATGGTTGCCGAGATCCTCGACGCGTGGGGGAAACCGATAGACCCGCGCGT

CGCGCACTGCATCTACGCCGGGTTGGCGACCGACACGGGGTCGTTTCGCTGGGCCAGTGTGCGG

GGGTATCGGCTGGCGGCGCGGCTGGTAGAGATCGGTGTGGACAACGCCACCGTCAGCAGGACC

TTGATGGACAGCCATCCCTTCACCTGGTTGCCGTTGCTATCGCGGGTGTTGGGTTCGGCGCAGCT

GGTGTCCGAGGCGGTCGGTGGCCGCGGGCTGGTTTACGTCGTCGTCGACAACCGGGAGTGGGTC

GCTGCGCGCTCGGAGGAAGTGGAAAGCATCGTCGACATCGTCCGCACCACGCAACAAGCCGAG

GTCGCGGCGGTGTTCAAGGAGGTCGAACCGCATCGGTGGTCGGTGTCGATGCGGGCTAAGACCG

TGAATTTGGCCGCGGTTGCCTCTGGGTTCGGTGGCGGTGGTCACCGGCTGGCCGCGGGGTATAC

GACCACCGGCTCGATCGACGACGCTGTGGCGTCGTTGCGCGCGGCGCTTGGTTAG

SEQ ID NO:16

Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP (also known as CnpB, rv2837c, or MT2903, 3 '-to-5' oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA) from M.tuberculosis, amino acid sequence (336 amino acids; M.tuberculosis protein WP _003905944.1 NCBI/GenBank reference sequence: AL123456, from M.tuberculosis strain H37 Rv. The M.tuberculosis protein has 20 additional amino acids at its N-terminus compared to the BCG protein (SEQ ID NO: 14), which are underlined and bolded.

Overexpression of CDN by knocking out endogenous BCG phosphodiesterase domain: the protein BCG _ RS07340 (formerly BCG _ 1416c) EAL Domain of (A). The BCG _ RS07340 protein (SEQ ID NO: 4) is encoded by the DNA sequence shown in SEQ ID NO: 5. The BCG _ RS07340 protein is 100% identical to mycobacterium tuberculosis Rv1354c protein and is an endogenous CDN PDE in BCG. The full-length polypeptide is 623 amino acids in length and it encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase. The domain structure is: N-terminal-GAF-GGDEF-EAL-C-terminal, as shown. The GAF domain (about amino acids 1-190) is a regulatory junction that affects the activity of other domainsAnd (4) domain formation. The GGDEF domain (about amino acids 190-350) is a diguanylate cyclase that catalyzes the reaction 2GTP → c-di-GMP +2 pyrophosphate. The EAL domain (amino acids 354 to 623, highlighted in SEQ ID NO: 4) is a diguanylate phosphodiesterase that catalyzes the reaction c-di-GMP → 2 GMP. Since the EAL domain of this protein is known to cleave 3'-5'c-di-GMPP, knocking out this endogenous cyclic dinucleotide phosphodiesterase domain will increase the level of c-di-GMP produced by BCG. Targeted knockout of the EAL domain can be accomplished by gene replacement of the full length WT BCG _ RS07340 gene with a gene encoding only amino acids 1-353 (GAF-GGDEF domain), i.e., the coding sequence of the gene is truncated to remove the sequence encoding amino acids 354-623 (as shown by the underlined DNA sequence in SEQ ID NO: 5) and to incorporate the appropriate stop codon and transcription termination sequence. Recombinant BCG lacking the EAL domain of BCG _ RS07340 will result in elevated CDN PAMP c-di-GMP levels.

Overexpression of CDN by knockout of endogenous BCG phosphodiesterase: BCG (BCG-computer-generated BCG) AHM07112。The BCG _ AHM07112 protein is the endogenous diguanylate phosphodiesterase in BCG (homologous to the 307 amino acid mycobacterium tuberculosis Rv1357c protein). Some BCG strains completely lack BCG _ AHM07112, while other BCG strains such as BCG Tice carry BCG _ AHM07112. In BCG strains with the polypeptide, the protein can be 288 amino acids in length (such as in BCG ATCC 35743) or 307 amino acids in length (such as in BCG Pasteur 1173P 2). The BCG AHM07112 protein from BCG ATCC35743 is 288 amino acids in length and 100% identical to mycobacterium tuberculosis Rv1357C protein over its C-terminal 287 amino acids. The domain structure of BCG _ AHM07112 is that of a single EAL domain. Since the M.tuberculosis Rv1357c protein is known to cleave 3'-5'c-di-GMP, it is highly likely that the BCG protein will undergo the same reaction. Knocking out this endogenous cyclic dinucleotide phosphodiesterase in BCG is expected to increase the level of c-di-GMP produced by BCG. Targeted knockout of the EAL domain can be accomplished by gene replacement of the full-length WT BCG _ AHM07112 gene and subsequent generation of a marker-free deletion.

SEQ ID NO:17

Diguanylate phosphodiesterases AHM07112.1, amino acid sequence (288 amino acids; genBank reference: CP003494.1; from BCG strain ATCC 35743) from BCG and other related Mycobacteria. AHM07112.1 is 100% identical to the C-terminal 287 amino acids of M.tuberculosis protein Rv1357C or MT1400 and the diguanylate phosphodiesterase of M.bovis, such as protein Mb 1392C.

MIDYEEMFRGAMQARAMVANPDQWADSDRDQVNTRHYLSTSMRVALDRGEFFLVYQPIIRLADNRIIGAE

ALLRWEHPTLGTLLPGRFIDRAENNGLMVPLTAFVLEQACRHVRSWRDHSTDPQPFVSVNVSASTICDPG

FLVLVEGVLGETGLPAHALQLELAEDARLSRDEKAVTRLQELSALGVGIAIDDFGIGFSSLAYLPRLPVD

VVKLGGKFIECLDGDIQARLANEQITRAMIDLGDKLGITVTAKLVESPSQAARLRAFGCKAAQGWHFAKA

LPVDFFRE

SEQ ID NO:18

Diguanylate phosphodiesterases AHM07112.1, DNA sequence (867 nucleotides [288 codons, 1 stop codon ]; genBank reference sequence: CP003494.1; from BCG strain ATCC 35743) from BCG and other related Mycobacteria. AHM07112.1 is 100% identical to the C-terminal 287 amino acids of M.tuberculosis proteins Rv1357C or MT1400 and the diguanylate phosphodiesterase of M.bovis such as protein Mb 1392C.

SEQ ID NO:19

Diguanylate phosphodiesterases Rv1357c or MT1400 from Mycobacterium tuberculosis and BCG Pasteur 1173P2, amino acid sequence (307 amino acids, NCBI/GenBank reference: AL123456; from Mycobacterium tuberculosis strain H37 Rv). An N-terminal extension of 19 amino acids was present in M.tuberculosis and BCG Pasteur strain 1173P2, but not in several other BCG strains. The 19 amino acid N-terminal extension is underlined and bolded. The C-terminal 287 amino acids of M.tuberculosis Rv1357C are 100% identical to BCG diguanylate phosphodiesterase AHM 07112.1.

The sequences cited in this application are summarized in table 1 below.

TABLE 1

In one embodiment, the invention relates to an expression cassette or vector comprising a nucleic acid sequence encoding an Rv1354c protein or functional portion thereof; a nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; or a combination thereof. In some aspects, the expression vector or cassette further comprises a nucleic acid sequence encoding a DNA integrity scanning (dis a) protein or functional portion thereof that functions as a diadenosine cyclase. In other aspects, the nucleic acid sequence encoding Rv1354c protein does not comprise a phosphodiesterase gene or a phosphodiesterase domain. In some aspects, the expression vector or expression cassette does not comprise a phosphodiesterase gene or a phosphodiesterase domain.

Methods for producing expression vectors and expression cassettes, transforming mycobacteria and isolating mycobacteria have been described. In some aspects, the expression vectors or expression cassettes of the invention comprise one or more regulatory sequences, such as promoter and/or enhancer elements, which control or influence the transcription of the nucleic acid, operably linked to the nucleic acids of the invention. In some aspects, an expression vector or expression cassette of the invention comprises one or more sequences operably linked to a nucleic acid of the invention that direct the termination of transcription, post-transcriptional cleavage, and/or polyadenylation. In some aspects, the expression vectors or expression cassettes of the invention comprise variable length intervening sequences and/or selectable marker genes operably linked to a nucleic acid of the invention.

In one embodiment, the invention relates to a mycobacterium strain comprising an expression vector or expression cassette of the invention described herein. In some aspects, the mycobacterium strain is mycobacterium tuberculosis, mycobacterium bovis, or a combination thereof. In other aspects, the mycobacterium strain is BCG. In some aspects, the strain comprises plasmid SEQ ID NO 13.

In another embodiment, the invention relates to mycobacterium strains that express or overexpress a diadenosine cyclase and/or that express or overexpress one or more other cyclase genes or domains (e.g., those described herein). In some aspects, 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 invention relates to mycobacterium strains that express or overexpress a diadenosine cyclase and/or do not express a Phosphodiesterase (PDE) that hydrolyzes STING agonists (e.g., a deletion of a PDE gene that contains a hydrolysis STING agonist). In some aspects, the mycobacterium strain is mycobacterium tuberculosis, mycobacterium bovis, or a combination thereof. In some aspects, the mycobacterium strain is BCG.

Statistically significant antitumor Effect of BCG-disA-OE in rat MNU bladder cancer model

The rat MNU bladder cancer model is a validated bladder cancer model in which intravesical administration of BCG can show therapeutic efficacy (figure 6 and Kates et al PMID 28588015). The inventors extended their previous findings on the therapeutic efficacy of BCG-disA-OE versus BCG-WT, shown in FIG. 7. The inventors have now performed the 16-week rat MNU model twice. FIG. 7 is based on experiment 1 and shows that BCG-disA-OE shows a trend towards better results than BCG-WT. After experiment 2 was performed and its data combined with experiment 1, BCG-dis a-OE was now shown to be statistically significantly better than no treatment (p = 0.048), while BCG-WT was not statistically significantly better than no treatment (data shown in fig. 15).

Tumor suppressor Treg cells were reduced by BCG-disA-OE in a murine syngeneic bladder cancer tumor model.

In the MNU rat bladder cancer model, the amount of bladder tissue at the end of the 16 week experiment was insufficient for flow cytometry. To investigate the cell population changes induced by BCG-disA-OE, a murine syngeneic bladder cancer tumor model was developed using BBN975 cells. This model allowed the development of large tumors (diameter >1.5 cm) on the flank of the mouse. Mice were treated with BCG-disA-OE and BCG-WT by intratumoral injection. As shown in figure 16, use of BCG-disA-OE resulted in decreased levels of tumor-associated CD4+ Treg cells, tumor-associated CD8+ Treg cells, and splenic CD4+ Treg cells.

Delivery of BCG-DISA-OE from intracellular compartment (intracellular compartment) for sustained STING agonism And (4) an agent.

Persistence of BCG in the bladder.

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

Durek et al, (The fat of bacillus calmeter-Guerin after intravenous infection. J Urol.2001; 165; 1765-1768.PMID 11342972) 49 patients were studied for continuous urine culture following intravesical BCG. BCG in urine was detected in 96.4% of the samples after 2 hours post-instillation, and BCG in urine was detected in 67.9% of the samples after 24 hours post-instillation. The number of positive samples decreased and was 27.1% on day 7 immediately before the next instillation (fig. 38). Researchers also assessed mycobacterial DNA by PCR for bladder biopsies within 1 week after the 6 th instillation (administered monthly instillation). Mycobacterial ribosomal DNA was found in 14 out of 44 bladder biopsies (31.8%). Furthermore, positive PCR of mycobacterial DNA was evident up to 24 months in between 4.2% and 37.5% of biopsies studied.

The fact that BCG is known to persist in bladder tissue represents an important advantage of the BCG-disA-OE strategy for delivering STING agonists in cancer. While many techniques focus on the generation of small molecule STING agonists, such agents have a relatively short exposure time. In contrast, BCG, as an intracellular microorganism and as demonstrated by the studies of Bowyer and Durek, persists in cells and tissues for several weeks. The persistence of BCG-disA-OE in tissues provides for sustained long-term delivery of STING agonists in the tumor microenvironment.

BCG-disA-OE is safer than BCG-WT

In humans, intravesical BCG treatment is associated with dysuria, fatigue, and weakness in the treated patients. Additional more serious adverse effects are persistent cystitis and disseminated BCG disease (BCGosis) of BCG. The patient safety of BCG was extensively reviewed in O' Donnell et al (as of 2019). The incidence of BCG dissemination into the bloodstream following intravesical instillation was estimated to be 1/15,000 patients.

To test the safety of BCG-disA-OE compared to BCG-WT, the inventors used two mouse models of BCG infection, in which BCG strains were aerosolized into the lungs of immunocompetent BABL/c mice or immunosuppressed SCID mice. As shown in fig. 17A-17B, BCG-disA-OE was less able to proliferate in immunocompetent mouse lungs than BCG-WT, and in immunosuppressed mice, BCG-disA-OE was less lethal in the time-to-death assay.

BCG elicitation training exemptions have been shownEpidemic, the training immunity and BCG in solid and liquid tumors and diabetes Is relevant. BCG strains overexpressing STING agonists elicit stronger changes in the training immunity than BCG-WT

And (4) training immunity.Training immunity refers to the ability of an antigenic stimulant to elicit a stronger immune response against a second, different antigen. Training immunity is antigen-independent, based on heterologous CD4 and CD8 memory activation, cytokine-mediated, and associated with epigenetic and metabolic changes. BCG is an effective tool as a first antigenic stimulator to elicit a training immunity to subsequent antigenic stimulations such as tumors, viral infections or drug-resistant bacterial infections (Neetea et al. Trained immunity: a program of input immune in health and disease. Science 2016.PMID 27102489; and Arts et al. BCG vaccination technologies against experimental viral infection in humans through the infection of cells associated with cultured immune in Host microorganism 2018.PMID 29324233).

BCG for solid and liquid tumors.BCG has a long history of therapeutic benefit as an immunotherapy for both solid and liquid tumours in humans (Hersh et al BCG as adjuvant immunotherapy for neoplasma. Annu Rev Med 1977.Pmid 324372). BCG has been used systemically and intratumorally in malignancies including melanoma, non-small cell lung cancer (NSCLC) and Acute Lymphoblastic Leukemia (ALL). There have recently been trials of BCG together with checkpoint inhibitors for various forms of bladder cancer.

BCG for diabetes.BCG vaccination has recently been shown to have therapeutic benefit on glycemic control in various forms of diabetes, including Type 1diabetes (Stienstra and net. Figure up diabetes: BCG vaccination effects on Type 1diabetes mellitis. Trends endothelial meta 2018. Pmid. This effect is thought to be mediated by the training immunity of BCG, which has been shown to result in epigenetic modifications that promote expression of pro-inflammatory cytokines and metabolic enzymes such as those used for glycolysisAnd (4) expressing.

BCG-disA-OE and training immunization.To investigate the ability of BCG strains overexpressing STING agonists to stimulate training immunity, the inventors tested that BCG-WT versus BCG-disA-OE elicited an increase in secondary antigen stimulation in resting human monocytes after six days of exposure to the BCG strain. The first antigen was the BCG strain at day 0 and after 6 days of rest the second antigen was the unrelated TLR-1/2 antigen PAM3CSK4. As can be observed in FIG. 18, the immune response (secretion of IL-1. Beta.) tested was enhanced by both BCG-WT and BCG-disA-OE after receiving the second stimulus, but the degree of stimulation by BCG-disA-OE was statistically significantly greater than the degree of stimulation without the first BCG stimulus or BCG-WT as the first stimulus. This revealed that BCG strains overexpressing STING, such as BCG-disA-OE, are more effective training immunostimulants than BCG-WT.

In related experiments, the present inventors performed the same BCG-first stimulation/6 days of rest/second antigen stimulation with TLR-1,2 of PAM3CSK4 with human monocytes. At the end of the experiment, cellular DNA was collected and chromatin immunoprecipitation (ChIP) was performed using antibodies against H3K4 histone methylation markers. The H3K4 marker is a known transcriptional activation marker. After quantitative PCR amplification of the IL-6 promoter region of the immunoprecipitated DNA, the results showed that BCG-Pasteur-dis A-OE and BCG-Tice-dis A-OE elicited the H3K4 marker in the IL-6 promoter (IL-6 is a proinflammatory cytokine) statistically significantly more efficiently than their respective BCG-WT strains. These results show that BCG strains overexpressing STING, such as BCG-disA-OE, are more potent stimulators of epigenetic changes associated with the training immunity than BCG-WT.

BCG-Tice-disAthe-OE gene expresses much higher levels of the disA gene than BCG-WT

As can be observed in fig. 21, use 2 ^-ΔΔCT Comparative methods, the relative expression of BCG-tie-dis-OE clone 2 (which was selected for seed lot preparation and storage) was 300. This indicates that the disA is strongly overexpressed by being on a multicopy plasmid and driven by the Mycobacterium leprae hsp65 promoter in the pSD5-hsp65-MT3692 plasmid.This strong overexpression resulted in much higher levels of release of the STING agonist c-di-AMP.

STING agonist overexpressing BCG strains such as BCG-disA-OE trigger signaling in more than one model system Pro-inflammatory changes in the pathways and cytokine secretion profile.

The inventors tested strains overexpressing STING agonists, such as BCG-disA-OE, in more than one model system to assess their relative ability to trigger proinflammatory cytokine changes. BCG-disA-OE was statistically significantly superior to BCG-WT in most of its tests. When the comparison was not statistically significant, BCG-disA-OE gave the stronger of the two responses.

FIG. 23 also shows that the increase in type 1 IFN secretion in both BCG-disA-OE and BCG-WT is STING-dependent.

In summary, BCG-disA-OE is a more potent stimulator of proinflammatory cytokine expression and proinflammatory pathway induction than BCG-WT.

The following table summarizes the data:

table 2:

a method of producing recombinant BCG without antibiotic gene cassettes overexpressing STING agonist biosynthesis genes.

The dis A overexpression plasmid pSD5-hsp65-MT3692 carries a Kan resistance gene cassette which confers resistance to the antibiotic kanamycin. The present inventors disclosed a method of generating recombinant BCG without antibiotic gene cassette overexpressing STING agonist biosynthesis gene.

The Mycobacterium genetic operon panCD codes for the biosynthetic genes panC (pantothenate-. Beta. -alanine ligase gene) and panD (aspartate 1-des)A carboxylase gene). The gene products PanC and PanD are also referred to as vitamin B ₅ (a B vitamin) is essential for the biosynthesis of pantothenic acid. Pantothenic acid is a water-soluble vitamin, and is an essential nutrient for mycobacteria such as BCG. Animals require pantothenic acid to synthesize coenzyme a (CoA) and to synthesize and metabolize proteins, carbohydrates, and fats. The anion is known as pantothenate.

It has been shown that the genetic deletion of panCD in mycobacteria results in mutant strains that can only grow in the presence of added pantothenate. Thus, the mutant strain is auxotrophic for pantothenate. The Δ panCD mutant of mycobacterium tuberculosis has been shown to be highly attenuated in animal infections, being rapidly cleared because it cannot grow in mammalian tissues where pantothenate is not available.

The inventors disclosed a detailed method for generating a marker-free (antibiotic-free gene cassette) Δ panCD deletion mutant of BCG. The mutant will only be able to grow in the presence of pantothenate and is not expected to survive during infection or to serve as an effective delivery vehicle for STING agonist expression.

The present inventors disclose a detailed method for generating a shuttle plasmid carrying the mycobacterial panCD gene and an overexpression construct for the biosynthesis of STING agonists (such as a Phsp65:: DISA construct which overexpresses the DISA gene and releases the excess STING agonist c-di-AMP). The shuttle plasmid is capable of replication in E.coli or Mycobacteria. It carries an antibiotic cassette that can be conveniently removed by cleavage with a rare-cutting restriction enzyme and religation. Alternatively, a shuttle plasmid may be generated by: the backbone of the PCR amplified plasmid without the antibiotic resistance cassette created restriction sites at the ends and ligated into the PCR product consisting of the amplified panCD operon with the same unique restriction sites at its ends. In either way, the shuttle plasmid (ligation product) without the antibiotic resistance gene can be electroporated into BCG or escherichia coli auxotrophs and selected on pantothenate-free agar plates.

In a final manifestation of the disclosure, the inventors show a method of introducing plasmids carrying antibiotic-free cassettes of the mycobacterial panCD gene and overexpression constructs for the biosynthesis of STING agonists (such as the Phsp65:: dis A construct) into the marker-free BCG Δ panCD mutants. The end result is a BCG strain that does not carry antibiotic resistance genes and that strongly overexpresses one or more than one STING agonist biosynthesis genes. In mammalian hosts or humans, such BCG strains will retain plasmids under strong selective pressure, as it requires panCD complementation from the plasmid.

In another manifestation of the disclosure, the panCD cassette and a construct for biosynthesis of STING agonists (such as a Phsp65:: disA construct) can be introduced into a chromosomal integration vector such as pMH 94. Using a similar approach, the antibiotic cassette could be eliminated from pMH 94. Introduction of this chromosomal integration plasmid into the unmarked BCG Δ panCD mutant also resulted in BCG strains that did not carry antibiotic resistance genes and strongly overexpressed one or more than one STING agonist biosynthesis genes. The disadvantage of this strategy is that the overexpression construct is single copy on the bacterial chromosome, rather than multiple copies on the plasmid, and this may result in lower levels of STING agonist release.

BCG-Tice (ATCC 35743) is a natural pantothenate auxotroph.

The present inventors have disclosed that the mycobacterium bovis BCG Tice strain (ATCC 35743) is a natural pantothenate auxotroph. This strain carries a 5bp DNA insertion in its panC gene at base pairs 739-743. This insertional mutational change results in a frame shift mutation after amino acid 246 of PanC (the length of wild-type PanC is 309 amino acids). Due to the 5bp insertion mutation, the mutant PanC polypeptide in the mycobacterium bovis BCG Tice strain (ATCC 35743) comprises 246 amino acids of the wild-type PanC sequence at its N-terminus followed by a nonsense polypeptide of 478 amino acids at its C-terminus. It is highly unlikely that the mutant PanC polypeptide will retain any functional pantothenate- β -alanine ligase activity (normal enzyme function of PanC). Furthermore, the panD polypeptide in BCG Tice (ATCC 35743) is very unlikely to be translated because the stop codon of the panC gene, which overlaps with the ATG for the initiation of translation of panD in the wild-type sequence, does not fit in frame (out of frame). In the wild-type panCD operon, ribosome termination of PanC translation is coupled with ribosome initiation of PanD translation. Since there is no ribosome termination immediately upstream of the panD start codon, ribosome initiation of panD gene translation is highly unlikely to occur.

The inventors disclose that this natural auxotrophy enables the more rapid construction of recombinant BCG without antibiotic gene cassettes that overexpress STING agonist biosynthetic genes.

The present inventors disclose a method for the direct introduction of plasmids carrying antibiotic-free cassettes of the mycobacterial panCD gene and overexpression constructs for the biosynthesis of STING agonists (such as the Phsp65:: dis A construct) into BCG-Tice (ATCC 35743).

pSD5-hsp60-MT3692 is identical to pSD5-hsp65-MT3692. The present inventors previously mentioned this same plasmid as pSD5-hsp60-MT3692. However, the actual promoter in this strain is the promoter of the hsp65 gene of Mycobacterium leprae. Therefore, the present inventors can refer to the plasmid pSD-hsp60-MT3692 as pSD5-hsp65-MT3692.

In one embodiment, the invention relates to a pharmaceutical composition comprising an expression vector, expression cassette or strain of the invention described herein and a pharmaceutically acceptable carrier.

In another embodiment, the present invention relates to a method and/or composition for treating and/or preventing cancer, comprising administering to a subject an expression vector, expression cassette, strain, or pharmaceutical composition described herein. 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 present disclosure contemplate methods and/or compositions for treating and/or preventing bladder cancer, wherein modulation of a type 1 Interferon (IFN) response is directly or indirectly related. In certain aspects, individuals having bladder cancer, such as NMIBC, are treated with a modulator of type 1 interferon response, and in some aspects, individuals having bladder cancer are provided with a modulator of type 1 interferon expression, such as an inducer of expression thereof.

In certain aspects, the level at which the type 1 interferon expression inducer increases the expression of type 1 interferon can be any level so long as it provides an improvement in at least one symptom of bladder cancer, including non-muscle invasive bladder cancer (NMIBC). In at least some cases, the expression level of type 1 interferon can be increased at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, or more compared to the expression level in a standard. The individual may monitor the expression level of type 1 interferon using standard methods in the art, such as, for example, RNA assay (northern assay) or quantitative PCR.

Individuals known to have, suspected of having, or at risk of having bladder cancer may be provided with an effective amount of an inducer of type 1 interferon expression, including the BCG strain of the invention containing the expression vector of the invention. The expression vector expresses RV1354c protein or a functional part thereof; a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a DNA integrity scanning (dis a) protein or functional part thereof that functions as an adenylate cyclase; or a combination thereof. It is preferred that the BCG strain of the present invention comprising the expression vector of the present invention is administered into the bladder of a subject, and the expressed one or more than one protein enhances the expression of type 1 interferon in the bladder. For example, those individuals at risk of bladder cancer may be individuals with one or more genetic factors, may be elderly individuals, and/or may have a family history.

In particular aspects of the disclosure, an individual is administered an agent for bladder cancer therapy in addition to one or more type 1 interferon inducers of the invention. Such additional therapies may include intravesical chemotherapy such as, for example, mitomycin C, cyclophosphamide, or a combination thereof. When a combination therapy is used with one or more type 1 interferon inducers, such as a strain of BCG expressing one or more of the following proteins.

In some aspects, the expression vectors, expression cassettes, strains, pharmaceutical compositions and/or methods of the invention described herein have increased safety, increased tolerance (e.g., reduced dysuria, urgency or weakness), and/or reduced likelihood of causing or disseminating bloodstream infections as compared to non-recombinant BCG.

In one embodiment, the present invention relates to a method of treating and/or preventing cancer comprising administering to a subject an expression vector, expression cassette, strain and/or pharmaceutical composition of the present invention described herein, wherein administration results in increased safety, increased tolerance (e.g., reduced dysuria, urgency or weakness), and/or a reduced likelihood of bloodstream or disseminated bloodstream infection as compared to non-recombinant BCG. In some aspects, the cancer is, e.g., 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.

In some embodiments, the present invention relates to a method of increasing safety, increasing tolerance (e.g., reducing dysuria, urgency, or weakness), and/or reducing the likelihood of causing or disseminating a blood stream infection as compared to non-recombinant BCG, comprising administering to a subject an expression vector, expression cassette, strain, and/or pharmaceutical composition of the invention described herein.

Pharmaceutical preparation

The pharmaceutical compositions of the invention comprise an effective amount of one or more type 1 interferon expression inducers, such as a BCG strain that expresses one or more of the following proteins, dissolved or dispersed in a pharmaceutically acceptable carrier: RV1354c protein or a functional portion thereof; a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a DNA integrity scanning (dis a) protein or functional part thereof that functions as an adenylate cyclase. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other abnormal reactions (untoward reactions) when administered to animals such as, for example, humans, as appropriate. In light of The present disclosure, the preparation of pharmaceutical compositions comprising at least one type 1 interferon expression inducer or additional active ingredients will be known to those skilled in The art, as exemplified by Remington: the Science and Practice of Pharmacy, 21 st edition Lippincott Williams and Wilkins,2005, which is incorporated herein by reference. Further, for animal (e.g., human) administration, it will be understood that the formulations should meet sterility, pyrogenicity, general safety and purity Standards as required by the FDA Office of Biological Standards.

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, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials, and combinations thereof, as will be known to those of ordinary skill in the art (see, e.g., remington's pharmaceutical Sciences, 18 th edition. Mack Printing Company,1990, pp.1289-1329, which is incorporated herein by reference). Except to the extent that any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

Type 1 interferon expression inducers (such as BCG strains expressing one or more of the following proteins: RV1354c protein or a functional part thereof; cyclic GMP-AMP synthase (DncV) protein or a functional part thereof; cyclic GMP-AMP synthase (cGAS) protein or a functional part thereof; DNA integrity scanning (dis) protein or a functional part thereof acting as adenylate cyclase) may comprise different types of vectors depending on whether it is administered in solid, liquid or aerosol (aerosol) form and whether it needs to be sterile in such administration routes as injection. In some aspects, the invention (e.g., expression vector, strain or Pharmaceutical composition) may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intravesically (e.g., directly into the bladder, such as by injection or by intravesical instillation), intratumorally, topically, intramuscularly, subcutaneously, mucosally, orally, topically (locally), by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, via a catheter, by cream, by direct local perfusion of bathing (localized bathing) target cells in liquid compositions (e.g., liposomes), or by other methods known to those of ordinary skill in the art or any combination of the foregoing (see, e.g., pharmaceutical's scientific, 18 th edition, mack Printing Company,1990, and human biological-systemic animal research applications, francica, incorporated by the study of the patent of the year 898 publication: STamino acids and methods).

Pharmaceutically acceptable salts include acid addition salts, for example, formed with free amino groups of the protein composition, or with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, or mandelic acid. Salts with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. After formulation, the solution will be administered in a manner compatible with the dosage formulation and in an amount such as is therapeutically effective. The formulations are readily administered in a variety of dosage forms, such as formulated for parenteral administration, such as an injectable solution or aerosol for delivery to the lung, or formulated for alimentary tract administration, such as a drug release capsule or the like.

Further, in accordance with the present disclosure, compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier should be absorbable and include liquid, semi-solid (i.e., paste) or solid carriers. Except to the extent that any conventional vehicle, agent, diluent or carrier is deleterious to the recipient or to the therapeutic effect of the composition contained therein, it is suitable for use in an administrable composition used in practicing the methods of the present invention. Examples of carriers or diluents include fats, oils, water, salt solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The composition may also comprise a plurality of antioxidants to retard oxidation of one or more components. In addition, 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., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

In accordance with the present invention, the compositions are combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, and the like. Such procedures are conventional to those skilled in the art.

In particular aspects of the invention, the composition is intimately combined or admixed with a semi-solid carrier or a solid carrier. The mixing may be carried out in any convenient manner, such as milling. Stabilizers may also be added during mixing 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, and the like.

In a further aspect, the invention includes the use of a pharmaceutical lipid vehicle composition comprising a type 1 interferon expression inducer, one or more lipids and an aqueous solvent. As used herein, the term "lipid" includes any of a wide range of substances that are characteristically insoluble in water and extractable with organic solvents. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are generally a biological substance. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids (lysolipids), glycosphingolipids, glycolipids, sulfates, lipids with ether and ester linked fatty acids, and polymerizable lipids, and combinations thereof. Of course, the compositions and methods of the present invention also encompass compounds other than those specifically described herein, which are understood by those of skill in the art to be lipids.

One of ordinary skill in the art will be familiar with the range of techniques that can be used to disperse the composition in a lipid vehicle. For example, the type 1 interferon expression inducers of the present invention may be dispersed in a solution containing a lipid, solubilized with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bound to a lipid, contained as a suspension in a lipid, contained or complexed with micelles or liposomes, or associated with a lipid or lipid structure by any means known to one of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

The actual dosage amount (dosage amount) of the composition of the invention to be administered to an animal patient may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic intervention, the patient's characteristics and the route of administration. Depending on the dosage and route of administration, the preferred dosage and/or the number of administrations of the effective amount may vary depending on the response of the subject. In any case, the physician responsible for administration will determine the concentration of the one or more active ingredients in the composition and the appropriate dose or doses for the individual subject.

In certain aspects, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other aspects, for example, the active compound can comprise between about 2% to about 75%, or between about 25% to about 60%, and any range derivable therein, by weight of the unit. Naturally, the amount of one or more active compounds in each therapeutically useful composition can be prepared in such a way that a suitable dose 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, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and thus, a variety of dosages and treatment regimens may be desirable.

In other non-limiting examples, the dose can also include 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 ranges derivable from the values listed herein, based on the values described above, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, and the like, may be administered.

A. Digestive Composition (Alimentary Composition) and formulation

In one embodiment of the disclosure, the type 1 interferon expression inducer of the invention is formulated for administration via the digestive route. The digestive route includes all possible routes of administration for which the composition is in direct contact with the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. Thus, these compositions may be formulated with an inert diluent or an ingestible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly into the food of the diet.

In certain aspects, the active compound may be incorporated into excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers (wafer) and the like (Mathiowitz E, jacob JS, jong YS, carono GP, chickering DE, chaturvedi P, santos CA, vijayaraghavan K, montgomery S, basett M, morrell c.biological edible microspheres aqueous powders patent oral Drug delivery systems. Nature.1997; 386-4. PMID 9121559, hwang MJ, ni X, waldman M, ewig CS, hagler AT.Derivation of class II for fields.VI.Carbohydrate compounds and anthroomeric effects.biopolymers.1998, 435-68.PMID. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as, for example, gum tragacanth, acacia, corn starch, gelatin or combinations thereof; excipients such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; a disintegrating agent such as, for example, corn starch, potato starch, alginic acid, or a combination thereof; lubricants, such as, for example, magnesium stearate; a sweetening agent such as, for example, sucrose, lactose, saccharin or combinations thereof; flavoring agents such as, for example, peppermint, oil of wintergreen, cherry flavoring (cherry flavoring), orange flavoring (orange flavoring), and the like. 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 example, 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, a carrier, such as a liquid carrier. Gelatin capsules, tablets or pills may be enteric coated. The enteric coating prevents denaturation of the composition in the stomach or upper bowel (upper bowel) where the pH is acidic. See, for example, U.S. patent No.5,629,001. Upon reaching the small intestine, the alkaline pH therein dissolves the coating and allows the composition to be released and absorbed by specialized cells, such as intestinal epithelial cells (epithelial enterocytes) and Peyer's patch M cells. A syrup or elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. Furthermore, the active compounds can be incorporated into sustained-release preparations (preparation) and preparations (formulation).

For oral administration, the compositions of the present disclosure may optionally incorporate one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, buccal spray, or sublingual oral formulation. For example, mouthwashes may be prepared by incorporating the required amount of active ingredient in a suitable solvent, such as a sodium borate solution (Dobell solution). Alternatively, the active ingredient may be incorporated into an oral solution, such as a solution containing sodium borate, glycerin, and potassium bicarbonate, or dispersed in a dentifrice, or added in therapeutically effective amounts to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and wetting agents. Alternatively, the composition may be formulated as a tablet or solution that can be placed under the tongue or otherwise dissolved in the mouth.

Additional formulations suitable for other modes of digestive administration include suppositories. Suppositories are solid dosage forms of varying weight and shape, usually filled with a drug, for insertion into the rectum. After insertion, the suppository softens, melts or dissolves in the luminal fluid. Generally, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain aspects, suppositories may be formed from mixtures containing the active ingredient in the range of, for example, about 0.5% to about 10%, and preferably about 1% to about 2%.

B. Parenteral compositions and formulations

In another embodiment, the type 1 interferon expression inducer of the present invention may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally. See, for example, U.S. patent nos. 6,7537,514; 6,613,308; U.S. Pat. No.5,466,468; U.S. Pat. No.5,543,158; U.S. Pat. No.5,641,515; and 5,399,363 (each of which is specifically incorporated by reference herein in its entirety).

Solutions of the active compound as a free base or pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No.5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that there is ease of injection. It must be stable under the conditions of manufacture and storage and must be protected from 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 can 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 dispersions and by the use of surfactants. Prevention of the action of microorganisms can be caused 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 of agents delaying absorption, for example, combinations of aluminum monostearate and gelatin.

For example, for parenteral administration as an aqueous solution, 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 particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used in accordance with the present disclosure will be known to those skilled in the art. For example, a dose may be dissolved in isotonic NaCl solution and added as a subcutaneous perfusion solution, or injected into the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences" 15 th edition, pages 1035-1038, and pages 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. In addition, for human administration, the formulations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA office of biological standards.

Sterile injectable solutions are prepared by: the desired amount of active compound is incorporated in a suitable solvent together with several of the other ingredients enumerated above, as required, and then filter sterilized. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains an alkaline 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 the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powdered composition is combined with a liquid carrier such as, for example, water or a salt solution, with or without a stabilizer.

C. Other pharmaceutical compositions and formulations

In some aspects of the invention, the active compounds of the invention (inducers of type 1 interferon expression) may be formulated for administration via a variety of other routes, such as topical (i.e. transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation. Pharmaceutical compositions for topical administration may comprise an active compound configured for drug-containing applications such as ointments, pastes, creams or powders. Ointments include all oily, adsorptive, emulsion and water-soluble based compositions for topical application, while creams and lotions are those containing only an emulsion base. Topically applied drugs may contain permeation enhancers to promote absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerol, alcohols, alkyl methyl sulfoxides, pyrrolidones, and laurocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorbent, lotion or water-soluble ointment base. Topical formulations may also include emulsifiers, gelling agents, and antimicrobial preservatives as needed to preserve the active ingredients and provide a homogeneous mixture. Transdermal administration of the present invention may also include the use of "patches". For example, the patch may provide one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

In certain embodiments, the pharmaceutical composition may be delivered by eye drops, intranasal sprays, inhalants, and/or other aerosol delivery vehicles. Methods of delivering compositions directly to the lungs via nasal aerosol sprays have been described, for example, in U.S. patent nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Similarly, drug delivery using intranasal microparticle resins (Takenaga M, serizawa Y, azechi Y, ochiai A, kosaka Y, igarashi R, mizushima Y. Microparticulate resins as a potential nasal drug delivery system for insulin. J Control Release.1998; 52. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene 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 solids of liquid particles dispersed in a liquefied or pressurized gaseous propellant. A typical aerosol for inhalation according to the invention will consist of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary depending on the pressure requirements of the propellant. The administration of the aerosol will vary depending on the age, weight and severity of symptoms and response of the subject.

Kits of the present disclosure

Any of the compositions described herein can be included in a kit. In a non-limiting example, a type 1 interferon expression inducer of the present invention (such as a BCG strain expressing one or more of the following proteins: RV1354c protein or a functional portion thereof; cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; DNA integrity scanning (dis) protein functioning as adenylate cyclase or a functional portion thereof) can be included in the kit.

The kit may comprise a suitable aliquot of an inducer of type 1 interferon expression of the invention, and in some cases, one or more additional agents. One or more components of the kit may be packaged in an aqueous medium or in lyophilized form. The container means of the kit will generally comprise at least one vial, test tube, flask, bottle, syringe or other container means in which the components may be placed and preferably suitably aliquoted. Where more than one component is present in the kit, the kit will typically further comprise a second container, a third container or other additional containers in which additional components may be separately placed. However, various combinations of components may be contained in the vial. The kit of the invention will also typically contain a means for containing an inducer of type 1 interferon expression of the invention and any other reagent containers within closed confines for commercial sale. Such containers may include injection or blow-molded plastic containers in which the desired vials are stored.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred. One or more type 1 interferon expression inducers of the compositions of the invention may be formulated into injectable compositions. In this case, the container means may itself be a syringe, pipette and/or other such similar device 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 other components of the kit.

However, the components of the kit may be provided as one or more dry powders. When the reagents and/or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent may also be provided in another container means.

The examples above have been included to provide guidance to those 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 in the art will appreciate that the above embodiments are intended to be exemplary only and that numerous changes, modifications and alterations may be used without departing from the scope of the presently disclosed subject matter. The above embodiments are provided by way of illustration and not by way of limitation.

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.

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.

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.

Presented below are examples discussing the enhancement of training immunity by redesigned BCG overexpressing the PAMP molecule cyclic di-AMP, envisaged for the application in question. The following examples are provided to further illustrate embodiments of the present invention and are not intended to limit the scope of the present invention. While these are typical of those that may be used, other procedures, methods, or techniques known to those skilled in the art may alternatively be used.

Examples

Example 1

Materials and methods

With particular reference to fig. 39-62, the following materials and methods are provided to illustrate in detail the methods for obtaining the presented results.

Bacterial strains and culture conditions：

M.bovis BCG (BCG) Pasteur (BCG-WT Pasteur) (Pink Collins Ph. FDA]Generous and the same as BCG-Pasteur provided to Trudeau research Institute at TMC No.1011 by the Pasteur research Institute (Pasteur Institute) 1967) and commercially available BCG-Tice (R) ((R)

Merck) was used to produce recombinant BCG strains overexpressing c-di-AMP. CDC1 from M.tuberculosis (M.tb) strain551 genomic DNA for PCR amplification of dis A (MT 3692/Rv 3586). Individual isolated bacterial colonies grown on 7H11 plates supplemented with oleic acid-albumin-dextrose-catalase (OADC) (cat.b11886, fisher Scientific) were picked and propagated in 7H9 midlebrook 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 the E.coli strain DH 5-alpha (Cat. 18258012, fisher Scientific) and maintained routinely in LB broth. To generate BCG overexpressing dis a, the mycobacterium tuberculosis gene MT3692 or Rv3586 was cloned under the strong mycobacterial promoter hsp60 using the e.coli-mycobacterium shuttle vector (psd 5.Hsp 60). Clones were confirmed by gene sequencing and used for bacterial transformation by electroporation. Recombinant strains were confirmed using colony PCR against kanamycin cassette and subjected to whole genome sequencing and qPCR analysis. Details of all bacterial strains, plasmids and constructs are listed in table 3.

Table 3:

mammalian cell culture:

cell line: for cell-based in vitro infection assays, J774.1 (American type culture Collection-

TIB67 ^TM Manassas, va., USA) murine macrophage cell line was enriched with 10% heat-inactivated Fetal Bovine Serum (FBS) (Cat.10082147, fischer Scientific) and 1% streptomycin/penicillin in RPMI-Glutamax (Cat.61870-036, fischer Scientific) at 37 ℃ and 5% CO ₂ And (5) culturing. Urothelial cancer cell line 5637 (

HTB-9 ^TM ) Human high grade urothelial cancer; RT4 (

HTB-2 ^TM ) Human transitional cell low-grade urothelial cancer; j82 (ii) (

HTB-1 ^TM ) Human high grade urothelial cancer; and NBT II (

CRL-1655 ^TM ) N-butyl-N- (4-hydroxybutyl) nitrosamine-induced tumor cell line No. 2 tumor of Nara bladder of Sus portus; UPPL1595 (luminal cell line established from spontaneous primary bladder tumors in Uroplakin-Cre driven PTEN/P53 knockout genetically engineered mouse models and generously provided by Philiam Kim doctor (UNC Chapel Hill)); BBN975 (basal cell line established from 0.05% N-butyl-N- (4-hydroxybutyl) nitrosamine (BBN) -induced murine urothelial cancer model and generously provided by William Kim doctor (UNC Chapel Hill)); and MB49 (from 7,12-dimethylbenzo [ a ]]Anthracene (DMBA) -induced murine urothelial carcinoma cells (EMD Millipore, cat. SSC148)) in RPMI 1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin at 37 ℃ and 5% CO ₂ Is maintained as a single layer. Mouse fibroblast cell line NCTC clone 929[ 2 ] L cell, L-929, derivative of L line](

CCL-1 ^TM ) At supplement with 1DMEM medium with 0% heat-inactivated Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin at 37 ℃ and 5% CO ₂ Is conventionally maintained in a single layer. All cell lines were maintained for no more than 10 subculture cycles and Mycoplasma (Mycoplasma) testing was performed periodically while the cells were in culture. The mouse cell line RAW-Lucia ISG (InvivoGen, calif., USA) was reported to be cultured in custom-prepared medium according to the manufacturer's instructions.

Primary cells (macrophages and dendritic cells):

to generate murine Bone Marrow Derived Macrophages (BMDM) and dendritic cells (BMDC), 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 (C57 BL/6J-Tmem173gt/J, jackson laboratories). More than one vial of bone marrow cells were stored in liquid nitrogen in cryopreservation media containing 10% DMSO (cat.d 2650; sigma) and 90% heat-inactivated FBS (cat.10082147, fischer Scientific). To differentiate BM cells into macrophages or DCs, random cryopreserved vials were selected and differentiated for 6 days in BMDM differentiation medium supplemented with 30% sterile mouse fibroblasts L929 (see: S.A.) and (see: S.B.) 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 vitamins (Cat. 11120052, thermo Fisher Scientific), and antibiotics (penicillin-streptomycin solution)

CCL-1 ^TM ) DMEM in conditioned medium. Differentiation of BM cells into DCs was performed in a low-attachment 10mm cell culture dish in the presence of recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (Cat. 315-03, peprotech) in the presence of bone marrow differentiation medium for 48h. Non-adherent cells were washed and loosely attached cells were allowed to differentiate into BMDCs for the next 6 days. Cells were characterized for macrophage and DC markers using cell surface staining and flow cytometry analysis. Human primary monocytes and human monocytesDerived Macrophages (HMDM) were used for cell-based in vitro infection assays. Peripheral blood-derived mononuclear cells (PBMCs) isolated from healthy male donors (leukocks) between the ages of 18 and 30 were used for isolation of Human Monocytes (HM) or human monocyte-derived macrophages (HMDM). For separating blood components and buffy coat, in Ficoll-Paque ^TM RPMI-1640 diluted blood was subjected to density gradient centrifugation (400 Xg at 18 ℃ for 30 min) on Plus reagent (Cat.17-1440-02, GE healthcare, piscataway, NJ). Cells were washed several times with 1xPBS and counted using a hemocytometer. After counting, CD14+ human monocytes were isolated from PBMC using magnetic labeling (monocyte isolation kit II, cat.130-091-153, miltenyi Biotec, san Diego, calif.) and a magnetic column according to the manufacturer's instructions. The purity of the isolated CD14+ cells was confirmed using cell fractions stained with a fluorochrome-conjugated antibody against a monocyte marker as recommended by the manufacturer, and the cells were analyzed using a BD-LSR2 flow cytometer. Inoculating human monocyte (2.0-3.0 × 10) ⁵ Individual cells/ml) in RPMI 1640 medium supplemented with 10% FBS and 1% streptomycin/penicillin at 37 ℃ and 5% CO ₂ . In the next 7 days, the CD14+ monocyte monolayer differentiated into M1[ GM-CSF (20 ng/ml, peproTech, rocky Hill, NJ) and IFN- γ (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)]。

Animals:

experimental procedures involving live animals were performed according to protocols approved by The Institutional Animal Care and Use Committee (IACUC) of The Johns Hopkins University School of Medicine. For animal infection protocols, pathogen-free 4-6 week old females 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 in animal biosafety grade 3 animal facilities without cross-ventilation. Fischer344 female rats (Harlan, average body weight 160 g) at 8 weeks of age were housed in a BSL2 animal facility. Animals were freely harvested for water and standard rat chow (standard chow) and general behavior and appearance was monitored daily by a veterinary specialist.

In vitro infection assay:

for in vitro infection assays, cell lines or primary cells were seeded at the desired cell density in 6-well tissue culture plates or 10mm culture dishes. For infection, log phase wild type strain and BCG-disA-OE strain were harvested by centrifugation and washed twice with DPBS to remove residual detergent and BSA, then suspended in antibiotic-free RPMI 1640 medium supplemented with 10% FBS. For infection assays, bacteria were deposited at a pre-calibrated multiplicity of infection (MOI). The infection was allowed to continue for the next 4 hours, and then the infected cells were washed repeatedly using warm DPBS to remove non-internalized bacteria. Infected cells were incubated in the presence of RPMI-1640 medium supplemented with 10% FBS and antibiotics until the endpoint.

And (3) toxicity determination:

human urothelial cancer cell lines RT4, 5637 and J82 in RPMI 1640 with 10% FBS and no antibiotics at 37 ℃ and at 5% CO ₂ Culturing under the condition. For cytotoxicity assays, 3000 RT4 cells and 1500 5637 and 1500J 82 cells were seeded in triplicate in 96-well tissue treatment plates, respectively. 24 hours after inoculation, cells were treated with the indicated BCG to cell ratio for 72 hours. To measure cell viability, cellTiter-Glo luminescent cell viability assay (Promega, madison, wis., USA) and FLUOstar OPTIMA (BMG Labtech, ortenberg, germany) were used according to the manufacturer's protocol. Relative cell viability was calculated by dividing the viability of the indicated ratio by the viability of the control.

For annexin-PI staining, 50 million J774.1 cells and BMDM were plated in each well of a 6-well plate for physical attachment. Wild-type strains of Tice and Pasteur and BCG-disA-OE strains were used to expose cells to 1. At the end of infection or treatment, cells were removed non-enzymatically using 0.02% EDTA-PBS solution. Cells were washed twice with ice-cold PBS and stained for FITC-annexin-PI using FITC annexin V apoptosis detection kit I (cat.556547, BD Biosciences) according to the manufacturer's instructions. Flow Cytometry was performed using The Bloomberg School of Public Health, johns Hopkins University Flow Cytometry BD LSR II Flow cytometer. Data were processed using FlowJo software (Tree Star v 10).

Quantitative real-time QPCR:

gene expression profiling was performed using total RNA isolated from cell lines or primary cells. For RNA isolation from rat bladder, whole bladder sample pieces were excised, snap frozen in liquid nitrogen immediately after harvest, and stored in RNAlater (cat. Am7021, ambion) at-80 ℃. Total RNA isolation was performed using the RNeasy system (Cat.74106, qiagen). Real-time qPCR was performed using the StepOnePlus system (Applied Biosystems). For gene expression analysis of cell lines and primary cells, SYBR Fast green double stranded DNA binding dye (Cat. 4085612, applied Biosystems) was used. Gene expression analysis of rat bladder tissue was performed using the TaqMan gene expression assay. Gene-specific qPCR primers were purchased from Integrated DNA Technologies, and all TaqMan gene expression assays were purchased from Thermo Fischer Scientific. Amplification of RNU6a, β -actin, GAPDH served as endogenous controls for RNA samples derived from human cells/tissues, mouse cells/tissues and rat cells/tissues, respectively. All experiments were performed in at least triplicate and data analysis was performed using the 2- Δ Δ CT method. Detailed information of NCBI gene identifiers and primer sequences is provided in table 4.

Table 4:

ELISA：

sandwich ELISA was performed to measure cytokines (IFN-. Gamma., TNF-. Alpha., IL-6, IFN-. Beta., IL-1. Beta. And MCP-1/CCl 2) in culture supernatants and animal tissues from lung, spleen or bladder. Tissue and culture supernatants were snap frozen in liquid nitrogen immediately after harvest and stored at-80 ℃. Animal tissues were homogenized using a microtissue homogenizer (cat. 1215d61, kimble) and filter sterilized to measure various cytokine protein expression levels using a sandwich ELISA according to the manufacturer's recommendations. Details of all ELISA kits and auxiliary reagents are provided in table 4.

Multi-color confocal microscopy:

multicolor laser confocal microscopy experiments were performed to determine phagocytosis, autophagy, and co-localization studies of urothelial cancer cells and primary macrophages. Cells were allowed to adhere to sterile glass coverslips placed in 6-well tissue culture plates and infected at a pre-calibrated MOI. Logarithmic phase bacterial cultures were labeled with FITC (cat. F7250, sigma). After infection and treatment conditions, cells were fixed, permeabilized and blocked, and then incubated overnight at 4 ℃ with the recommended dilutions of primary antibody to LC3B (Cat. NB100-2220, novus) or p62/SQSTM1 (Cat. P0067, sigma-Aldrich). Cells were washed and incubated with Alexa Flour 647 conjugated secondary antibody (cat. A32733, thermo Fisher Scientific) in the dark for 1 hour at 4 ℃. DNA staining was performed for 5min using Hoechst33342 (Cat.62249, thermo Fisher Scientific). Images were acquired at Microscope Facility, johns Hopkins School of Medicine using a Zeiss LSM700 single point laser scanning confocal Microscope at 63 magnification. Image processing and analysis was performed using open source Fiji software. For LC3B or p62 quantification, perinuclear LC3B spots (puncta, spot) were counted in at least 100 cells across different fields using Imaris 9.5.0. Quantification was performed using GraphPad Prism software.

Phagocytosis assay:

an IgG-FITC conjugated latex bead phagocytosis assay kit (product number 500290, cayman Chemicals, USA) was used for phagocytosis studies. HMDM was placed on a sterile glass coverslip for attachment. Infection was performed at a rate of 5:1 (HMDM to BCG) for 3 hours, followed by addition of IgG-FITC beads diluted in 1. Nuclear staining was performed using Hoechst33342 (cat.62249, thermo Scientific) and bead phagocytosis of cells was visualized using Zeiss LSM700 single point laser scanning confocal microscope. Quantification of beads was measured by calculating the mean fluorescence intensity (m.f.i.) using open source Fiji software.

Multicolor flow cytometry:

cell surface and intracellular staining was performed on J774.1, murine BMDM, human HMDM, and single cells derived from murine MB49 tumor and spleen. Flow cytometry groups were designed and, if desired, modified forms of murine and lymphoid myeloid and human myeloid cells. Details of all antibodies and dilutions used are provided in table 3. For in vitro infection assays, protein transport inhibitor mixtures (Cat. 00-4980-03, eBioscience) were added at the recommended dilutions 12 hours prior to harvesting of the cell monolayers. At the end point, cells were harvested using cell detachment buffer (ice cold PBS-10mM EDTA solution). Single cell isolation was performed using animal tissue by harvesting tumors and spleens after necropsy. Briefly, tissues were manually disrupted and then incubated in RMPI for 30min at 37 ℃ in collagenase I (Gibco) and DNase (Roche). Tumor and splenocytes were separated by 70 μm filter and washed with PBS. RBC lysis was performed using ACK lysis buffer (cat. A1049201, thermo Fisher Scientific) for 5 minutes at room temperature. Cells were washed twice with ice-cold PBS and with Zombie Aqua ^TM Staining with Fixable viatility kit (Cat.423101, biolegend). Cells were washed and resuspended in FACS buffer (1% BSA, 2mM EDTA in PBS) and Fc blocked (TruStain FcX) according to the manufacturer's protocol ^TM Cat.101320 and True-Stain monoclonal packer ^TM Cat.426102biolegend) and staining with conjugated primary antibody. Intracellular staining was performed after immobilization and permeabilization (immobilization and permeabilization buffer set, eBioscience). Cells were washed and resuspended in flow buffer (flow buffer) and fed using BD LSRII with FACSDiva softwareAnd (5) line acquisition. Analysis was performed using FlowJo (v 10) (TreeStar).

The following antibodies were used to stain myeloid and lymphoid lineage cells:

mouse BMDM: anti-CD 45 (clone 30-F11), anti-CD 124 (clone I015F 8), anti-I-A/I-E (clone 107630), anti-LY 6C (clone HK 1.4), anti-CD 11b (clone M1/70), anti-F4/80 (clone BM 8), anti-Ly 6G (clone 1A 8), anti-CD 206 (clone C068C 2), anti-TNF (clone MP6-XT 22) are all Biolegend, and anti-IL-10 (clone JES5-16E3 eBiosciences).

Human HMDM: anti-CD 16 (clone 3G 8), anti-CD 14 (clone 63D 3), anti-HLA-DR (clone L243), anti-CD 11b (clone ICRF 44), anti-CD 206 (clone 15-2), anti-CD 163 (clone GHI/61), anti-TNF (clone MAb 11) and anti-TNF (clone MAb 11) were all Biolegend.

Mouse macrophages (urothelial carcinoma syngeneic MB49 model): CD45 (clone 30-F11, biolegend), CD124 (IL-4 Ra) (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 JES 5-16983, thermo Fisher).

Mouse T cells (urothelial carcinoma syngeneic MB49 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-. Gamma. (clone XMG1.2, biolegend) and FOXP3 (clone MF-14, biolegend).

In vitro monocyte training immune experiment:

in vitro training of primary human monocytes was performed 47 as described previously. PBMCs were isolated from healthy donors (leukappak). After magnetic separation, CD14+ monocytes were seeded in warm RPMI 1640 medium supplemented with 10% FBS in 10mm3 tissue culture dishes at 37 ℃ and 5% ₂ For 3 hours. Non-adherent cells were removed by washing the cells with warm PBS. Infection of monolayer cultures of human monocytes with BCG-WT strain and BCG-disA-OE strain at a MOI of 5:1 (monocyte to BCG) in the presence of RPMI 1640 supplemented with 10% FBS for 4 hours. Non-internalized bacilli were washed out using warm PBS and then incubated for 24 hours. The cells were washed again with warm PBS and fresh warm RPMI 1640 medium was added. In the next 5 days, the cells were allowed to rest, washed with PBS and fresh medium was added every 2 days. Cells were restimulated on day 6 with RPMI 1640 supplemented with 10% FBS (negative control, untrained) or the TLR1/2 agonist Pam3Cys (cat. Tlrl-pms, invivoGen). After 24h of stimulation, culture supernatants were collected, filter sterilized and flash frozen (-80 ℃) for cytokine measurements. Cells were harvested for chromatin immunoprecipitation (ChIP) experiments to measure epigenetic changes on gene promoters.

Chromatin immunoprecipitation (ChIP): human monocytes were fixed with formaldehyde at a final concentration of 1% for 10 min at room temperature. Cell fixation was stopped using 125mM glycine (Cat No.50046, sigma-Aldrich, USA) followed by sonication using a Qsonica Sonicator Q125 (Cat.15338283, thermo Fisher Scientific) to fragment cellular DNA to an average size between 300bp and 600 bp. By mixing at 4 ℃ magnetic Dynabeads (Cat No.10004D, thermo Fisher Scientific, USA) with the recommended concentration of primary antibody [ (histone H3K9me3 (H3K 9 trimethyl) polyclonal antibody Cat. A-4036-100, epigentek); anti-histone H3 (trimethyl K4) antibody-ChIP grade (ab 8580), abcam) ] were incubated overnight and the sonicated cell lysates were subjected to Immunoprecipitation (IP). Unbound material was removed by washing the Dynabeads sequentially with lysis buffer, chromatin IP (ChIP) wash buffer and Tris-EDTA (TE buffer). DNA elution was performed using ChIP elution buffer. Different segments of the immune gene regulatory region were amplified with qPCR using specific primers. The reactions were normalized to the input DNA, while the beads served as negative controls. Details of all primary antibody and primer sequences are provided in table 4.

Targeted metabolite analysis with LC-MS/MS:

the targeted metabolite analysis was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS) as previously described 48. Metabolites from cells or flash-frozen xenograft tumor tissue were extracted with 80% (v/v) methanol solution equilibrated at-80 ℃ and the supernatant containing the metabolites was dried under nitrogen. The dried samples were resuspended in 50% (v/v) acetonitrile solution and 4ml of each sample was injected on a 5500QTRAP triple quadrupole mass spectrometer (AB Sciex) coupled to a Prominence Ultra Fast Liquid Chromatography (UFLC) system (Shimadzu) and analyzed. The instrument was operated in a Selective Reaction Monitoring (SRM) with positive and negative ion switching modes, as described. This targeted metabolomics approach allows the analysis of more than 200 metabolites from a single 25min LC-MS acquisition with 3MS residence time, covering all major metabolic pathways. The optimized MS parameters are: the ESI voltage is +5,000V in positive ion mode and-4,500V in negative ion mode; the dwell time was 3ms/SRM transition and the total cycle time was 1.57s. Hydrophilic interaction chromatography (HILIC) separations were performed on a Shimadzu UFLC system using an Amide column (Waters XBidge BEH Amide,2.1x 150mm,2.5 μm). The LC parameters are as follows: the column temperature is 40 ℃; the flow rate was 0.30ml/min. Solvent a, water containing 0.1% formic acid; solvent B, acetonitrile containing 0.1% formic acid; a non-linear gradient from 99% B to 45% B in 25 minutes and a post-run time of 5min. Peak integration of each targeted metabolite in SRM conversion was treated with multisant software (V2.1, AB Sciex). The preprocessed data on integrated peak areas were exported from the MultiQuant and reintroduced into the metamalyst software for further data analysis, including statistical and principal component analysis.

Histological analysis and Immunohistochemistry (IHC):

for histological analysis, a portion of the bladder was formalin-fixed and paraffin-embedded. Sections of 5 μ thick on the slides were stained with hematoxylin-eosin to classify according to World Health Organization/International urinary Pathology (World Health Organization/International Society of Urological Pathology) consensus as previously described 27. Tumor staging was performed by 2 urogenital pathologists (a.s.b., a.m.) certified by the committee. Samples were classified according to the percentage of abnormal tissue involvement (1 =10% involvement, 2=20% involvement, and so on). For IHC staining, high temperature antigen retrieval (18-23 psi/126 ℃) was performed by dipping the slides into Trilogy (Cell Marque). Endogenous peroxidase activity was blocked for 5min using a Dual endogenesis Enzyme Block (Cat. S2003, dako). The primary antibodies used included Ki67 (1, 50, cat. Ab16667, abcam), CD68 (1, 250, cat. Mcaherr), CD86 (1, cat.bs-1035r, bioss) and CD206 (1. For Ki67, sections were stained with ImmPACT DAB (Vector Labs) for 3min and counterstained with hematoxylin (Richard-Allen). Double staining of CD68/CD206 and CD68/CD86 was achieved by: CD68 was first stained with Impact DAB (Vector Labs), then secondary antigen retrieval was performed and incubated with CD86 or CD206 as described above and visualized with ImmPACT AEC (Vector Labs). For each section, ki67 expression was scored as the percentage of positive cells in the urothelium. Double staining for CD68/CD86 and CD68/CD206 was scored based on positive cell clusters for each marker (0 = no staining, 1= rare segregating cell positive, 2= cluster of up to 10 positive cells, 3= > cluster of 10 positive cells).

In vivo experiments:

intravesical BCG treatment in a carcinogen-induced NMIBC rat model:

induction of urothelial carcinoma in rats and subsequent intravesical BCG treatment was performed. N-methyl-N-nitrosourea (MNU) instillations were given every other week for 4 instillations. 7-week-old Fischer344 female rats (Harlan, average body weight 160 g) were anesthetized with 3% isoflurane. After complete anesthesia, a 20G vascular catheter was placed into the urethra of the rat. MNU (1.5 mg/kg) (Spectrum) dissolved in 0.9% sodium chloride was then instilled and the catheter removed and sedation continued for 60 minutes to prevent spontaneous urination and to allow absorption. PBS or 5X10 weekly administration 18 weeks after the first MNU instillation ⁶ Various BCG strains of CFU (0.3 ml through 20G vascular catheters) were treated intravesically for a total of 6 doses. Rodents were sacrificed 2 days after the last intravesical treatment and the bladders harvested within 48 hours after the last BCG instillation for mRNA and protein expression analysis and histological evaluation.

BCG infection and CFU count in BALB/c mice:

to determine the pulmonary bacterial load of the wild type strain and the BCG-disA-OE strain, 6-week-old female BALB/c mice were exposed using the aerosol route in the Glasscol inhalation exposure system (Glasscol). The inoculum implanted into the lungs of female BALB/c mice on day 1 (n =3 mice/group) was determined by plating whole lung homogenates on 7H11 selective plates containing carbenicillin (50 mg/ml), trimethoprim (20 mg/ml), polymyxin B (25 mg/ml) and actinone (10 mg/ml). After infection, mouse lungs (n =5 animals/group) were harvested, homogenized in sterile PBS in their entirety, and plated at different dilutions on 7H11 selective plates. The 7H11 selective plates were incubated at 37 ℃ and single colonies were counted at weeks 3 and 4. Single colonies were expressed as log CFU per organ.

SCID mouse death time study:

as previously described, virulence tests of BCG-WT and BCG-dis-A-OE strains were performed in a severely compromised immunodeficient mouse aerosol infection model. The inoculum implanted into the lungs on day 1 was determined by plating whole lung homogenates on 7H11 selective plates (n =3 animals/group). For time to death analysis (n =10 animals/group), infected animals were monitored until they died.

Syngeneic MB49 model of urothelial cancer:

the MB49 tumor cells were a urothelial cancer line obtained from adult C57BL/6 mice by: exposure of Primary urothelial cell explants to 7,12-dimethylbenzo [ a]Anthracene (DMBA) for 24 hours, followed by long-term culture 79. Prior to implantation, MB49 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% streptomycin/penicillin at 37 ℃ and 5% CO ₂ Culture was performed as a monolayer. Cells were harvested using trypsinization and cell viability was determined using trypan blue dye. Live MB49 cells were resuspended in sterile PBS and adjusted to 1X 10 ⁵ Viable cells/100. Mu.l. 4-6 weeks old female C57BL/6J mice (Charles River Laboratories) were injected subcutaneously 1X 10 on the right side of the hind leg ⁵ Individual MB49 cells. Tumor growth was monitored every 2 days to see the increase in tumor burden at the beginning of treatment. Palpable tumors (7 to 9 days, average volume-30 mm) ³ ) Thereafter, intratumoral injection of a total of 50. Mu.l of 1X 10 in PBS ⁶ BCG-WT bacilli or BCG-disA-OE bacilli (FIG. 41). 4 total BCG intratumoral injections are given once every 3 days. Tumors were measured by electronic calipers and tumor volume was calculated using the following equation: tumor volume = length × width × height × 0.5326. Mice were sacrificed at the indicated times and tumors and spleens were collected for single cell preparation after necropsy.

Example 2

BCG-DISA-OE elicits a higher macrophage pro-inflammatory cytokine response than BCG-WT.

BCG-DISA-OE is a genetically engineered BCG strain in which the endogenous polyadenylation cyclase gene, DISA, is fused to a strong promoter, resulting in 300-fold overexpression of DISA and 15-fold increase in the production of cyclic di-AMP (FIG. 39). BCG-disA-OE activated the STING pathway in macrophages significantly increased compared to BCG-WT, as measured by IRF3 induction (fig. 39). To test the fact that a number of BCG strains are used worldwide and their variability in clinical efficacy has been described, two forms of BCG-disA-OE and the corresponding BCG-WT were generated: one used BCG-dice, and one used BCG-Pasteur. No significant difference between the Tice and Pasteur forms was detected.

To characterize the training immune induction potential of BCG-disA-OE and BCG-WT, their ability to induce cytokine expression in human monocyte-derived macrophages (HMDM), primary murine bone marrow-derived macrophages (BMDM) and dendritic cells (BMDC), and macrophage cell line (J774.1) was evaluated. Consistent induction of IRF3, IFN- β, TNF- α and IL-6 in response to BCG-disA-OE was found in all myeloid cell types, which was significantly higher than that observed for BCG-WT exposed cells (fig. 40 and 43), and this difference was observed in human MDM and murine BMDM, even when the cells were primed with IFN- γ (fig. 43). These differences are strictly STING-dependent, e.g. using the signals from STING ^-/- BMDM of mice was confirmed (fig. 40). Since STING activation leads to upregulation of NF- κ B via the TBK1-IRF3 pathway, it was found that both TNF- α and IL-6 expression was similar to IFN- β expression in cells of the same panel and was significantly higher after exposure to BCG-disA-OE compared to BCG-WT (fig. 41 and fig. 43). Cyclic dinucleotides, including cyclic di-AMP, are known to be several chemokines (CXCL 9, CXCL10[ IP-10 ]]CXCL22 and MCP-1) anda potent inducer of iNOS; in agreement with this, IFN- γ -primed BMDM showed a stronger induction of these chemokines and iNOS when challenged with BCG-disA-OE strain compared to BCG-WT (FIG. 41). Cytotoxicity was also assessed using annexin-PI staining and it was found that while late apoptotic cell death remained at baseline for BCG-disA-OE exposure in both BMDM and J774.1 macrophages, BCG-WT exposure caused significantly higher levels of apoptotic cell death in BMDM cells (fig. 44). These observations indicate that BCG-disA-OE elicits more potent pro-inflammatory cytokine expression than BCG-WT in primary human MDM as well as murine primary macrophages and macrophage lines.

Example 3

Proinflammatory polarization of macrophages is greater for BCG-DISA-OE than for BCG-WT

Training immunity is associated with the polarization of macrophages to an inflammatory phenotype, with a concomitant shift from an anti-inflammatory state. To study macrophage polarization, flow cytometry was used to monitor the phenotypic shift of both murine and human primary macrophages after 24h exposure to BCG-disA-OE or BCG-WT. First, following in vitro BCG exposure, MHC class II expressing CD45 was focused using a gating protocol ⁺ CD11b ⁺ F4/80 ⁺ Murine BMDM population. As can be observed in FIGS. 50 and 48, significantly greater TNF-. Alpha.expressing CD11b was observed after exposure to BCG-disA-OE than for BCG-WT ⁺ F4/80 ⁺ (M1) amplification of murine BMDM. Next, for CD45 ⁺ CD11b ⁺ F4/80 ⁺ Expression of the M2 surface receptor CD206 in macrophages ⁺ And CD124 ⁺ Was gated and a greater reduction in this population was observed for BCG-disA-OE than for BCG-WT (FIGS. 50 and 49). In this immunosuppressive cell population, there is a higher proportion of IL-10 expressing CD206 in BCG-WT exposed macrophages ⁺ CD124 ⁺ Cells, whereas IL-10 expressing cells in response to BCG-dis a-OE exposure were significantly reduced (fig. 50 and fig. 49). These results indicate that BCG-disA-OE exposure results in more extensive macrophage reprogramming, and pro-inflammatory M1 macrophage amplification, shows increased antigen presentation (MHC class II expression) and TNF- α expression, and expresses IL-1 compared to BCG-WT0, reduction of immunosuppressive M2 macrophages.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells known to promote immunosuppression. Thus, monocyte-myeloid derived suppressor cell M-MDSC (CD 45) was studied using primary murine BMDM using the gating protocol shown ⁺ Ly6C ^hi Ly6G ^- CD11b ⁺ F4/80 ^- ) Induction of (4). Significant amplification of M-MDSCs was observed after BCG-WT exposure, whereas in contrast, this same population showed minimal amplification after BCG-disA-OE exposure (fig. 50). Furthermore, M-MDSC elicited by BCG-WT showed higher IL-10 expression, whereas there was almost no IL-10 expressing M-MDSC after BCG-disA-OE exposure (FIG. 50). These observations indicate that BCG-WT contributes to the amplification of M-MDSC with immunosuppressive properties; however, overexpression of the pro-inflammatory PAMP cyclic di-AMP by BCG prevented M-MDSC amplification.

Next, macrophage activation phenotype in HMDM isolated from several independent healthy human donors was characterized. Both the BCG-WT strain and the BCG-disA-OE strain give rise to the classical macrophage population (CD 11 b) ⁺ CD14 ⁺ CD16 ^- ) These induction in response to BCG-dis a-OE was relatively higher (fig. 51 and fig. 52). Classically activated antigen-presenting macrophages (CD 14) were examined ⁺ CD16 ^- HLA-DR ⁺ ) And their ability to produce TNF-alpha or IL-6, and have been found to produce HLA-DR of TNF-alpha and IL6 upon exposure to BCG-dis-A-OE as compared to BCG-WT ⁺ The proportion of cells was significantly increased (fig. 52 and 53). Also in transient or intermediate macrophages (CD 11 b) ⁺ CD14 ⁺ CD16 ⁺ ) The M2 surface marker CD206 was studied ⁺ And CD163 ⁺ And they were found to have a greater reduction in consistency after BCG-disA-OE exposure than for BCG-WT (FIG. 51, FIG. 53). The fraction of these intermediate macrophages expressing M2 surface markers and IL-10 was also significantly lower than BCG-WT in response to BCG-disA-OE exposure (FIG. 53). In summary, using both mouse and human primary macrophage ex vivo models, it was found that BCG-disA-OE promotes greater activation of macrophages towards the M1 phenotype (inflammatory) and at the same time reduces immunosuppression compared to BCG-WTCompetent cells included the presence of M-MDSC.

Example 4

Macrophages exposed to BCG-DISA-OE are more phagocytic than those exposed to BCG-WT

Cyclic dinucleotides are reported to recruit inflammatory macrophages that exhibit high phagocytic potential. Consistent with these observations, we demonstrate that HMDM transfected with cyclic di-AMP shows enhanced phagocytosis and exhibits elongated dendrites compared to mock-transfected populations. The phagocytic properties of HMDM after exposure to different BCG strains were then evaluated and it was found that macrophages exposed to BCG-disA-OE engulfed IgG-opsonized FITC-latex beads significantly more than BCG-WT (figure 54). Consistent with the previously established role of STING pathway activation in enhancing autophagy, it was found that most intracellular BCG-disA-OE bacilli co-localized with LC3B in IFN- γ activated primary BMDM, whereas autophagy induction in BCG-WT was significantly lower. It was also found that the co-localization of BCG-disA-OE bacilli with the autophagy adaptor p62 was significantly higher than that observed with BCG-WT. These results reveal that BCG-disA-OE increases the level of phagocytic and autophagic processing within macrophages to a greater extent than BCG-WT, a phenomenon associated with enhanced presentation of peptide antigens to MHC class II molecules.

Example 5

BCG-DISA-OE causes macrophage epigenetic reprogramming and enhances training immunity to a greater extent than BCG-WT

Given recent data showing that BCG is a potent inducer of training immunity through epigenetic modification of key pro-inflammatory genes, it was hypothesized that the addition of cyclic di-AMP overexpression in standard BCG might enhance epigenetic modification in primary human monocytes. It has been established that BCG-disA-OE is a more potent inducer of macrophage TNF- α and IL-6 secretion than BCG-WT, as demonstrated in primary human monocytes from a group of 6 healthy human subjects. The ability of traditional BCG to elicit training immunity is associated with changes in epigenetic signatures that increase expression of pro-inflammatory genes. Thus, it was asked whether the enhanced induction of TNF-. Alpha.and IL-6 expression by BCG-disA-OE was epigenetically mediated compared to BCG-WT. To this end, persistent, antigen-independent epigenetic changes in the promoter regions of the TNF-a and IL-6 genes were evaluated using an assay in which human monocytes exposed to BCG strain 24h were allowed to rest for 5 days and then challenged with the heterologous antigen TLR1/2 agonist Pam3CSK4 on day 6 (figure 56). The activated histone methylation marker H3K4me3 present in TNF-alpha and IL-6 promoters was quantified using a chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assay. It was observed that exposure to BCG-disA-OE resulted in a higher enrichment of this marker than BCG-WT, even in the absence of a heterologous secondary stimulation (i.e., addition of RPMI medium alone on day 6). After restimulation with Pam3CSK4 on day 6, both BCG strains further increased the abundance of the activated epigenetic marker, but BCG-disA-OE-pretreatment produced significantly more enrichment than BCG-WT (fig. 56). Similarly, the chromatin suppression marker H3K9me3 on the same two promoters was studied and it was found that although both BCG strains resulted in a decrease in H3K9me3 levels (which was further exacerbated by the addition of Pam3CSK 4), the extent of BCG-disA-OE-mediated decrease was consistently greater than the BCG-WT-mediated decrease after initial exposure and after resting and restimulation (fig. 56). Simultaneous measurement of TNF- α and IL-6 in BCG-trained culture supernatants after non-specific stimulation by Pam3CSK4 revealed that BCG-disA-OE-trained macrophages produced significantly higher levels of these proinflammatory cytokines compared to BCG-WT-trained macrophages. These results indicate that enhanced BCG overexpressing the PAMP molecule cyclic di-AMP results in significantly stronger epigenetic changes normally associated with the training immunity.

Example 6

BCG-DISA-OE causes reprogramming of macrophage immune metabolic status to pro-inflammatory features to a greater extent than BCG-WT

BCG training reportedly stimulates glycolysis and the tricarboxylic acid cycle through glutamine supplementation and fumaric acid accumulation. To address whether the addition of cyclic di-AMP overexpression would alter BCG-mediated metabolomic shift, LC-MS was used to characterize key metabolites in primary human macrophages and primary murine macrophages exposed to both BCG strains. HMDM or BMDM showed an increase in catabolic profile (elevated intracellular glucose and lactate) to a higher extent after 24h exposure to BCG-disA-OE compared to BCG-WT. Furthermore, the TCA cycle metabolites of BCG-disA-OE, itaconic acid and fumaric acid, were also increased more than BCG-WT. These observations indicate that carbon substrates for ATP production are more catabolized in BCG-disA-OE infected macrophages than infected BCG-WT, consistent with the pro-inflammatory bioenergy spectrum.

Tryptophan dehydrogenase and indoleamine 2,3-dioxygenase (IDO) catabolize excess tryptophan to kynurenine is intimately involved in immune suppression, and IDO inhibitors have shown potential as immune activators in a variety of infectious and neoplastic diseases. Kynurenine levels in macrophages after BCG-disA-OE exposure were significantly lower than those observed after BCG-WT exposure, and as expected, BCG-disA-OE resulted in elevated tryptophan levels, while BCG-WT resulted in tryptophan levels comparable to the baseline observed for heat-killed BCG controls. Citrulline levels of BCG-disA-OE are also higher than BCG-WT, while putrescine levels are lower than BCG-WT, indicating that BCG-disA-OE more strongly induces nitric oxide synthase-mediated conversion of arginine to NO (pro-inflammatory) and citrulline. Finally, it is interesting that BCG-disA-OE more efficiently induces the production of the isocitrate lyase inhibitor itaconic acid, shown to have antibacterial activity, by macrophages than BCG-WT. Thus, BCG-disA-OE elicits greater pro-inflammatory metabolomic profiles with reduced kynurenine accumulation and increased production of glycolytic metabolites, NOS products and itaconic acid, as compared to BCG-WT.

Example 7

In vivo functional efficacy: BCG-DISA-OE shows better immunotherapy results in relevant immune animal models for training

In addition to its use as a TB vaccine, BCG has been used as a first-line immunotherapy for the treatment of non-muscle invasive bladder cancer (NMIBC) since the mid 70's of the 20 th century. Recent studies have shown that BCG exerts its anti-tumor effects through a mechanism of trained immunity. It has been shown that boosting BCG with excess cyclic di-AMP release results in improved training immune parameters in a series of in vitro assays seeking to determine whether these effects can be manifested in vivo.

First, BCG-disA-OE and BCG-WT were tested in the carcinogen-induced NMIBC model, where intravesical therapy could be introduced into the bladder as it would in humans with non-invasive urothelial cancer. The rat N-methyl-N-nitrosourea (MNU) model of Bladder Cancer (BC) is graphed in fig. 57. In this model, urothelial dysplasia occurs 14 weeks after the first intravesical instillation of MNU, and by 24 weeks, rats exhibit different forms of urothelial cancer severity, including Carcinoma In Situ (CIS), papillary Ta (superficial) or higher T1-T2 urothelial cancer, and histopathological and immunophenotypic characteristics similar to those observed in human bladder cancer. After carcinogen-mediated tumor induction with 4 cycles of MNU (week 0, week 2, week 4, week 6), groups of rats were treated with 6 week doses of intravesical BCG-disA-OE, BCG-WT or untreated at weeks 18-23. At week 24 sacrifice, the rat bladder was divided in half for (i) RT-PCR analysis, and (ii) histological analysis, including tumor staging by a blinded urogenital pathologist. Transcriptional analysis of the entire excised bladder at week 24 showed that BCG-disA-OE caused significant increases in IFN- β, IFN- γ, TNF- α, IL-1 β, CXCL10, MCP-1, MIP-1 α and iNOS transcription levels compared to BCG-WT, while both BCG strains reduced the mRNA levels of the immunosuppressive cytokines IL-10 and TGF- β (FIG. 57). The pattern of expression of these cytokines was confirmed at the protein level using ELISA for TNF- α, IL-2 and IFN- γ, and it was noted that intravesical BCG-DISA-OE strongly increased IFN- γ levels in the spleen of rats, whereas BCG-WT did not. Accordingly, the highest pathological grade, tumor involvement index and highest tumor stage were found to be significantly reduced in rats treated with BCG-disA-OE compared to untreated (fig. 57). The tumor involvement index of BCG-disA-OE was statistically significantly superior to that of no treatment (p < 0.001) and BCG-WT (p < 0.05), whereas BCG-WT showed only an improved trend towards being superior to that of no treatment. Importantly, the highest tumor stage observed in BCG-disA-OE treated rats was CIS, whereas it was T1 in rats receiving BCG-WT and T2 in untreated rats, and 53.3% of BCG-disA-OE treated rats were cancer free (p = 0.009), compared to 31.2% of BCG-WT and 0% of untreated rats (fig. 57). Immunohistochemical analysis revealed a significant reduction of Ki67 staining in the bladder of BCG-disA-OE treated MNU rats when compared to untreated (p < 0.01) and BCG-WT (p < 0.05), indicating reduced tumor proliferation. CD68 staining of rat bladder showed significantly higher macrophage recruitment levels and a trend towards an increase in pro-inflammatory M1-like CD86+ macrophages, as well as a significant reduction in CD206+ M2-like macrophages associated with tumor promotion in BCG-disA-OE treated rats compared to untreated controls. These observations indicate that the induction of type I IFN and other pro-inflammatory features in the bladder of tumor-bearing rats treated with BCG-disA-OE correlates with an increase in the anti-tumor activity of the recombinant BCG strain.

The functional efficacy of BCG-disA-OE was also tested in a murine ectopic syngeneic bladder cancer model using MB49 urothelial cancer cells. After intraflank implantation of MB49 tumor cells, mice received 4 intratumoral treatments within 9 days, as shown in fig. 61. In this model, BCG-disA-OE also showed a stronger immunotherapeutic efficacy than BCG-WT, as measured by tumor volume and weight compared to BCG-WT after intratumoral injection of BCG-disA-OE (FIG. 61). Histopathology in MB49 tumors treated with BCG-disA-OE showed extensive necrosis and hyperemia when compared to BCG-WT and no treatment. The body weight of mice receiving BCG did not change significantly, however both BCG strains significantly increased spleen weight. We further characterized the effect of treatment on macrophage polarization and recruitment of activated T cells in the Tumor Microenvironment (TME). As shown in FIG. 61, BCG-disA-OE significantly reduced the abundance of immunosuppressive M2 macrophages when compared to untreated and BCG-WT, and significantly (p)<0.01 ) increases pro-inflammatory M1 macrophages. Similarly, BCG-disA-OE recruits significantly more IFN-. Gamma.producing CD4 when compared to BCG-WT ⁺ T cells, and both BCG strains produce IFN-gamma producing CD8 ⁺ T cells are increased. Although both BCG strains recruited more CD4 to tumors ⁺ Cells and CD8 ⁺ Cells, but BCG-disA-OE uniquely targeted to the spleen of treated animalsMore CD8 was recruited ⁺ T cells. BCG-disA-OE also reduced tumor-associated T regulatory (Treg) cells to a significantly greater extent than BCG-WT in both tumor and spleen. Consistent with our previous findings in primary cells, we also found that BCG-disA-OE elicited stronger cytokine responses and autophagy in human urothelial cancer cells representing different tumor stages compared to BCG-WT. These results indicate that BCG-disA-OE has superior anti-tumor efficacy to BCG-WT in this mouse model of urothelial cancer, and that its efficacy is associated with the polarization shift of macrophages to M1, CD4 ⁺ T cells and CD8 ⁺ Increased activation of both T cells and a reduction in local intratumoral and systemic Treg cell populations are associated.

Example 8

Safety: the pathogenicity of BCG-DISA-OE in two mouse models is lower than that of BCG-WT

To address the concern that the enhanced pro-inflammatory immune response elicited by BCG-disA-OE may lead to adverse effects, safety in two different mouse models was evaluated. An aerosol-exposed immunocompetent BALB/c mouse model was used and lung bacterial load was measured after four weeks when the adaptive immune response was maximal (fig. 58). Although the day 1 engraftment of the two BCG strains was equivalent, we observed that BCG-disA-OE proliferated significantly less in the mouse lungs than BCG-WT by a difference of 0.43log ₁₀ Colony forming units (FIG. 58). As previously observed in cell-based models, proinflammatory cytokine levels in both lung and spleen were significantly higher in mice exposed to BCG-disA-OE than in mice receiving BCG-WT (figure 60). Two strains were also tested in immunocompromised SCID mice that failed to survive BCG infection. Again, using the low dose aerosol exposure model (FIG. 59), a statistically significant prolongation of survival of BCG-disA-OE compared to BCG-WT was observed. Thus, BCG-disA-OE is less pathogenic than BCG-WT in both murine model systems, although it elicits more pronounced inflammatory features in many of the model systems.

Example 9

Discussion of the related Art

Many recombinant BCG strains have been produced and tested for many years. These studies are usually targeted at improving TB protection efficacy or bladder cancer immunotherapy, but in some cases, the goal is to prevent other infectious diseases. Common strategies are overexpression of antigens to elicit disease-specific immunity or overexpression of cytokine genes to enhance local host responses. Although many modified BCG showed efficacy in preclinical models, little progress has been made to human clinical trials. To date, only BCG Δ ureC:hly (VPM 1002), a BCG designed to enhance phagosomal permeability and exposure of BCG antigens to processing of cytoplasmic MHC class I antigens, has entered late clinical trials in tuberculosis. This was the first specific re-engineered BCG specifically aimed at improving the training immunity by increasing the involvement of the STING pathway by overexpressing the PAMP molecule cyclic di-AMP.

To determine whether the training immune parameters could be increased, BCG-disA-OE and BCG-WT were tested in a series of in vitro assays. Cytokine release profiles, macrophage polarization, autophagy, phagocytosis, epigenetic modification and metabolic remodeling in human and murine primary cells were evaluated. BCG-disA-OE is a more potent pro-inflammatory response enhancer than BCG-WT in each assay system. BCG expressing cyclic di-AMP was further tested in a functional in vivo assay for training immunity (i.e., bladder cancer immunotherapy). BCG-disA-OE had greater immunotherapeutic potency than BCG-WT in two different urothelial cancer models, suggesting that our in vitro results predict functional potency in relevant animal models. Interestingly, BCG-disA-OE did not produce excessive virulence in both animal models of BCG infection or BCGosis, despite the significantly stronger pro-inflammatory response elicited in our in vitro assay system.

It was also observed that BCG-WT did not consistently elicit pro-inflammatory responses. For example, it was observed that murine macrophages treated with BCG-WT actually induced a higher percentage of M-MDSC (anti-inflammatory) than untreated controls (figure 50), and similarly BCG-WT resulted in elevated levels of the anti-inflammatory metabolite kynurenine. These findings of certain anti-inflammatory effects of BCG-WT may be correlated with the following observations: in countries where BCG is routinely used for TB prevention, vaccinees show reduced levels of asthma and atopic dermatitis. In contrast, this amplification of M-MDSC in macrophages by BCG-WT was reversed by cyclic di-AMP overexpression, consistent with recent studies showing that activation of the STING pathway reduces MDSC induction in certain cancers.

The training immune changes elicited by BCG may underlie the immunotherapeutic role of BCG in cancer prevention. Therefore, another objective of this study was to assess whether the beneficial effects of BCG-disA-OE as NMIBC immunotherapy were mediated by participation in the STING pathway and modulation of BCG-mediated training immunity. In the rat model of NMIBC, it was found that although invasive tumors occurred in untreated tumor-bearing rats (highest tumor grade T2) as well as in BCG-WT treated animals (highest tumor grade T1), there was no invasive bladder cancer at all in BCG-disA-OE treated rats. Similarly, BCG-disA-OE outperformed BCG-WT in reducing tumor growth and associated increased tumor necrosis in the MB49 mouse model of bladder cancer, and these effects were accompanied by significantly higher recruitment of M1 macrophages, IFN- γ producing CD4 cells, and reduced accumulation of Treg cells in the tumor. Increased levels of proinflammatory cytokines and chemokines were observed in the bladder from tumor-bearing animals treated with BCG-disA-OE compared to BCG-WT. Since non-immune cells also appear to have immunological memory, the possibility that this cytokine response may originate in the myeloid lineage cells in the TME and/or the tumor cells themselves is considered. Indeed, it was found that BCG-disA-OE elicited a stronger cytokine response in primary macrophages and human urothelial cancer cells representing various tumor stages than BCG-WT. This is shown as a downstream result of STING activation, as we found it to be from STING ^-/- Expression in BMDM was significantly reduced in mice. In addition, strong induction of several chemokines was also found, as observed in other studies stimulated with exogenous STING agonists.

Although the present invention has been described with reference to the above examples, it is to be understood that modifications and variations are included within the spirit and scope of the present invention. Accordingly, the invention is not limited except as by the appended claims.

Although the present invention has been described with reference to the above examples, it should be understood that modifications and variations are included within the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Sequence listing

<110> university of John Hopkins

W.R. Bise

Cui Nidi. J. Bivalakua

A Luoke. Singh

Mo Nali. Prahalaj

Takashi Yoshida

<120> recombinant therapeutic intervention for cancer

<130> JHU4280-2WO

<140>

<141>

<150> 16/790,161

<151> 2020-02-13

<160> 107

<170> PatentIn version 3.5

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acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt 2700

gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac 2760

aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg 2820

tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg 2880

aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc 2940

aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca 3000

tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag 3060

ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt 3120

cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg 3180

cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa 3240

tcgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact 3300

gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta 3360

acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt 3420

gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt 3480

caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt 3540

ctggctggat gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg 3600

cagacctcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc 3660

agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt 3720

gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc 3780

ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac 3840

ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga 3900

caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag 3960

ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt 4020

taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt 4080

ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc 4140

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac 4200

cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt 4260

taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg 4320

ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg 4380

ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag 4440

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 4500

aaaaggatct tcacctagat ccttttaaaa gtgctcatca ttggaaaacg ttcttcgggg 4560

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 4620

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 4680

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 4740

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 4800

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 4860

ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 4920

acgaggccct ttcgtcttca agaattccca ggcatcaaat aaaacgaaag gctcagtcga 4980

aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 5040

atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 5100

gcccgccata aactgccagg gaattcccat cgagccgaga acgttatcga agttggtcat 5160

gtgtaatccc ctcgtttgaa ctttggatta agcgtagata cacccttgga caagccagtt 5220

ggattcggag acaagcaaat tcagccttaa aaagggcgag gccctgcggt ggtggaacac 5280

cgcagggcct ctaaccgctc gacgcgctgc accaaccagc ccgcgaacgg ctggcagcca 5340

gcgtaaggcg cggctcatcg ggcggcgttc gccacgatgt cctgcacttc gagccaagcc 5400

tcgaacacct gctggtgtgc acgactcacc cggttgttga caccgcgcgc ggccgtgcgg 5460

gctcggtggg gcggctctgt cgcccttgcc agcgtgagta gcgcgtacct cacctcgccc 5520

aacaggtcgc acacagccga ttcgtacgcc ataaagccag gtgagcccac cagctccgta 5580

agttcgggcg ctgtgtggct cgtacccgcg cattcaggcg gcagggggtc taacgggtct 5640

aaggcggcgt gtacgcggcc acagcggctc tcagcggccc ggaaacgtcc tcgaaacgac 5700

gcatgtgttc ctcctggttg gtacaggtgg ttgggggtgc tcggctgtcg cggttgttcc 5760

accaccaggg ctcgacggga gagcggggga gtgtgcagtt gtggggtggc ccctcagcga 5820

aatatctgac ttggagctcg tgtcggacca tacaccggtg attaatcgtg gtctactacc 5880

aagcgtgagc cacgtcgccg acgaatttga gcagctctgg ctgccgtact ggccgctggc 5940

aagcgacgat ctgctcgagg ggatctaccg ccaaagccgc gcgtcggccc taggccgccg 6000

gtacatcgag gcgaacccaa cagcgctggc aaacctgctg gtcgtggacg tagaccatcc 6060

agacgcagcg ctccgagcgc tcagcgcccg ggggtcccat ccgctgccca acgcgatcgt 6120

gggcaatcgc gccaacggcc acgcacacgc agtgtgggca ctcaacgccc ctgttccacg 6180

caccgaatac gcgcggcgta agccgctcgc atacatggcg gcgtgcgccg aaggccttcg 6240

gcggccgtcg acggcgaccg cagttactca ggcctcatga ccaaaaaccc cggccacatc 6300

gcctgggaaa cggaatggct ccactcagat ctctacacac tcagccacat cgaggccgag 6360

ctcggcgcga acatgccacc gccgcgctgg cgtcagcaga ccacgtacaa agcggctccg 6420

acgccgctag ggcggaattg cgcactgttc gattccgtca ggttgtgggc ctatcgtccc 6480

gccctcatgc ggatctacct gccgacccgg aacgtggacg gactcggccg cgcgatctat 6540

gccgagtgcc acgcgcgaaa cgccgaattc ccgtgcaacg acgtgtgtcc cggaccgcta 6600

ccggacagcg aggtccgcgc catcgccaac agcatttggc gttggatcac aaccaagtcg 6660

cgcatttggg cggacgggat cgtggtctac gaggccacac tcagtgcgcg ccagtcggcc 6720

atctcgcgga agggcgcagc agcgcgcacg gcggcgagca cagttgcgcg gcgcgcaaag 6780

tccgcgtcag ccatggaggc attgctatga gcgacggcta cagcgacggc tacagcgacg 6840

gctacaaccg gcagccgact gtccgcaaaa agccgtgacg cgccgaaggc gctcgaatca 6900

ccggactatc cgaacgccac gtcgtccggc tcgtggcgca ggaacgcagc gagtggctcg 6960

ccgagcaggc tgcacgcgcg cgaagcatcc gcgcctatca cgacgacgag ggccactctt 7020

ggccgcaaac ggccaaacat ttcgggctgc atctggacac cgttaagcga ctcggctatc 7080

gggcgaggaa agagcgtgcg gcagaacagg aagcggctca aaaggcccac aacgaagccg 7140

acaatccacc gctgttctaa cgcaattggg gacgggtgtc gcgggggttc cgtggggggt 7200

tccgttgcaa cgggtcggac aggtaaaagt cctggtagac gctagttttc tggtttgggc 7260

catgcctgtc tcgttgcgtg tttcgttgcg ccgttttgaa taccagccag acgagacggg 7320

gttctacgaa tcttggtcga taccaagcca tttccgctga atatcgggga gctcaccgcc 7380

agaatcggtg gttgtggtga tgtacgtggc gaactccgtt gtagtgcctg tggtggcatc 7440

cgtggccact ctcgttgcac ggttcgttgt gccgttacag gccccgttga cagctcaccg 7500

aacgtagtta aaacatgctg gtcaaactag gtttaccaac gatacgagtc agctcatcta 7560

gggccagttc taggcgttgt tcgttgcgcg gttcgttgcg catgtttcgt gtggttgcta 7620

gatggctccg caaccacacg cttcgaggtt gagtgcttcc agcacgggcg cgatccagaa 7680

gaacttcgtc gtgcgactgt cctcgttggg atctagcccg cctaatgagc gggctttttt 7740

tt 7742

<210> 4

<211> 623

<212> PRT

<213> Unknown (Unknown)

<220>

<223> description unknown: bifunctional diguanylate cyclase/phosphodiesterase sequences

<400> 4

Met Cys Asn Asp Thr Ala Thr Pro Gln Leu Glu Glu Leu Val Thr Thr

1 5 10 15

Val Ala Asn Gln Leu Met Thr Val Asp Ala Ala Thr Ser Ala Glu Val

20 25 30

Ser Gln Arg Val Leu Ala Tyr Leu Val Glu Gln Leu Gly Val Asp Val

35 40 45

Ser Phe Leu Arg His Asn Asp Arg Asp Arg Arg Ala Thr Arg Leu Val

50 55 60

Ala Glu Trp Pro Pro Arg Leu Asn Ile Pro Asp Pro Asp Pro Leu Arg

65 70 75 80

Leu Ile Tyr Phe Ala Asp Ala Asp Pro Val Phe Ala Leu Cys Glu His

85 90 95

Ala Lys Glu Pro Leu Val Phe Arg Pro Glu Pro Ala Thr Glu Asp Tyr

100 105 110

Gln Arg Leu Ile Glu Glu Ala Arg Gly Val Pro Val Thr Ser Ala Ala

115 120 125

Ala Val Pro Leu Val Ser Gly Glu Ile Thr Thr Gly Leu Leu Gly Phe

130 135 140

Ile Lys Phe Gly Asp Arg Lys Trp His Glu Ala Glu Leu Asn Ala Leu

145 150 155 160

Met Thr Ile Ala Thr Leu Phe Ala Gln Val Gln Ala Arg Val Ala Ala

165 170 175

Glu Ala Arg Leu Arg Tyr Leu Ala Asp His Asp Asp Leu Thr Gly Leu

180 185 190

His Asn Arg Arg Ala Leu Leu Gln His Leu Asp Gln Arg Leu Ala Pro

195 200 205

Gly Gln Pro Gly Pro Val Ala Ala Leu Phe Leu Asp Leu Asp Arg Leu

210 215 220

Lys Ala Ile Asn Asp Tyr Leu Gly His Ala Ala Gly Asp Gln Phe Ile

225 230 235 240

His Val Phe Ala Gln Arg Ile Gly Asp Ala Leu Val Gly Glu Ser Leu

245 250 255

Ile Ala Arg Leu Gly Gly Asp Glu Phe Val Leu Ile Pro Ala Ser Pro

260 265 270

Met Ser Ala Asp Ala Ala Gln Pro Leu Ala Glu Arg Leu Arg Asp Gln

275 280 285

Leu Lys Asp His Val Ala Ile Gly Gly Glu Val Leu Thr Arg Thr Val

290 295 300

Ser Ile Gly Val Ala Ser Gly Thr Pro Gly Gln His Thr Pro Ser Asp

305 310 315 320

Leu Leu Arg Arg Ala Asp Gln Ala Ala Leu Ala Ala Lys His Ala Gly

325 330 335

Gly Asp Ser Val Ala Ile Phe Thr Ala Asp Met Ser Val Ser Gly Glu

340 345 350

Leu Arg Asn Asp Ile Glu Leu His Leu Arg Arg Gly Ile Glu Ser Asp

355 360 365

Ala Leu Arg Leu Val Tyr Leu Pro Glu Val Asp Leu Arg Thr Gly Asp

370 375 380

Ile Val Gly Thr Glu Ala Leu Val Arg Trp Gln His Pro Thr Arg Gly

385 390 395 400

Leu Leu Ala Pro Gly Cys Phe Ile Pro Val Ala Glu Ser Ile Asn Leu

405 410 415

Ala Gly Glu Leu Asp Arg Trp Val Leu Arg Arg Ala Cys Asn Glu Phe

420 425 430

Ser Glu Trp Gln Ser Ala Gly Leu Gly His Asp Ala Leu Leu Arg Ile

435 440 445

Asn Val Ser Ala Gly Gln Leu Val Thr Gly Gly Phe Val Asp Phe Val

450 455 460

Ala Asp Thr Ile Gly Gln His Gly Leu Asp Ala Ser Ser Val Cys Leu

465 470 475 480

Glu Ile Thr Glu Asn Val Val Val Gln Asp Leu His Thr Ala Arg Ala

485 490 495

Thr Leu Ala Arg Leu Lys Glu Val Gly Val His Ile Ala Ile Asp Asp

500 505 510

Phe Gly Thr Gly Tyr Ser Ala Ile Ser Leu Leu Gln Thr Leu Pro Ile

515 520 525

Asp Thr Leu Lys Ile Asp Lys Thr Phe Val Arg Gln Leu Gly Thr Asn

530 535 540

Thr Ser Asp Leu Val Ile Val Arg Gly Ile Met Thr Leu Ala Glu Gly

545 550 555 560

Phe Gln Leu Asp Val Val Ala Glu Gly Val Glu Thr Glu Ala Ala Ala

565 570 575

Arg Ile Leu Leu Asp Gln Arg Cys Tyr Arg Ala Gln Gly Phe Leu Phe

580 585 590

Ser Arg Pro Val Pro Gly Glu Ala Met Arg His Met Leu Ser Ala Arg

595 600 605

Arg Leu Pro Pro Thr Cys Ile Pro Ala Thr Asp Pro Ala Leu Ser

610 615 620

<210> 5

<211> 1872

<212> DNA

<213> Unknown (Unknown)

<220>

<223> description unknown: bifunctional diguanylate cyclase/phosphodiesterase sequences

<400> 5

atgtgcaacg acaccgcgac gccgcagctt gaggagctcg tcaccaccgt agccaaccag 60

ctcatgacag tcgacgctgc cacgtcagcc gaagtcagtc agcgcgtttt ggcctatcta 120

gtggaacagc tgggcgtaga tgtcagcttt ttgcgtcata acgatcgcga caggcgcgcg 180

acgaggctgg tggccgaatg gccacctcgc ctcaacatac cggaccccga tccgctcagg 240

ctgatctact tcgctgatgc cgacccggtg tttgcgctat gcgaacacgc caaagagcct 300

ctcgtgttcc ggcccgagcc ggccaccgag gactatcaac gcctcatcga agaagcccgc 360

ggggttccgg taacgtcggc tgccgccgtg ccgctggtat ctggcgagat caccactgga 420

ctgctggggt tcatcaagtt cggtgatcgg aaatggcacg aggccgagct taacgccctc 480

atgaccatcg ctacactctt cgcccaggtg caggctcgcg tcgccgccga ggcgcggctt 540

cgctatctgg ccgaccatga cgatctgacc ggactgcata accgtcgcgc gttgctgcag 600

cacctggacc aaagactggc ccccggacaa cctggcccgg tcgcggcgct atttctcgac 660

ttggaccgcc tcaaggccat caacgactac ctgggccacg ccgccggtga ccagttcatc 720

catgtgttcg cccaacggat cggtgacgca ctcgttggcg agagcctgat cgcccgactc 780

ggcggcgacg aattcgtcct catacccgca tctccaatga gtgccgatgc cgctcaaccg 840

ctcgccgaac gtcttcgcga ccagctcaag gaccacgtcg ctatcggcgg tgaggtgctc 900

acccgcaccg tcagtatcgg tgtcgcctca gggactcccg gacagcacac accgtcggac 960

ctcctgcgcc gagccgacca agccgctctg gcagccaaac acgccggcgg agatagcgtc 1020

gcgattttca ccgcggacat gtcggtcagc ggcgaactgc gcaacgatat tgaactacac 1080

cttcgacgtg gtatcgaatc cgacgccctt cgcctggtct acctacccga ggtcgaccta 1140

cggaccggcg acattgtcgg gaccgaggca ttggtccggt ggcagcaccc cacccgtggg 1200

ctgctggcac cgggctgctt catccctgtg gccgaatcca tcaaccttgc aggcgaattg 1260

gatagatggg tgctgcggag ggcctgcaat gaattctccg agtggcagtc agccggtttg 1320

ggccacgacg cgctgctgcg tatcaacgtc tcagctggac agctggtgac gggcgggttt 1380

gttgacttcg tcgcagacac gatcggccag cacggtctgg acgcctcgtc cgtgtgtttg 1440

gaaatcaccg aaaacgttgt ggtgcaagac ctacataccg ccagagccac cctggctcga 1500

ctcaaagaag tcggcgttca catcgctatc gacgatttcg gcaccggcta tagcgccata 1560

tcactgttgc agacgctacc gatcgacacg ctcaagatcg acaaaacatt cgtgcggcaa 1620

ctcggaacca acactagcga tctggtcatt gtgcgcggca tcatgacact cgccgaaggc 1680

ttccaactcg atgtagtagc cgaaggcgtc gagaccgagg ctgccgccag aattctattg 1740

gatcagcgct gttaccgtgc gcaaggcttc ttgttctccc ggcctgtccc cggggaggcc 1800

atgcggcaca tgttgtccgc acgacgacta ccgccgacct gcatacctgc aactgacccg 1860

gcgttatctt ga 1872

<210> 6

<211> 353

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 6

Met Cys Asn Asp Thr Ala Thr Pro Gln Leu Glu Glu Leu Val Thr Thr

1 5 10 15

Val Ala Asn Gln Leu Met Thr Val Asp Ala Ala Thr Ser Ala Glu Val

20 25 30

Ser Gln Arg Val Leu Ala Tyr Leu Val Glu Gln Leu Gly Val Asp Val

35 40 45

Ser Phe Leu Arg His Asn Asp Arg Asp Arg Arg Ala Thr Arg Leu Val

50 55 60

Ala Glu Trp Pro Pro Arg Leu Asn Ile Pro Asp Pro Asp Pro Leu Arg

65 70 75 80

Leu Ile Tyr Phe Ala Asp Ala Asp Pro Val Phe Ala Leu Cys Glu His

85 90 95

Ala Lys Glu Pro Leu Val Phe Arg Pro Glu Pro Ala Thr Glu Asp Tyr

100 105 110

Gln Arg Leu Ile Glu Glu Ala Arg Gly Val Pro Val Thr Ser Ala Ala

115 120 125

Ala Val Pro Leu Val Ser Gly Glu Ile Thr Thr Gly Leu Leu Gly Phe

130 135 140

Ile Lys Phe Gly Asp Arg Lys Trp His Glu Ala Glu Leu Asn Ala Leu

145 150 155 160

Met Thr Ile Ala Thr Leu Phe Ala Gln Val Gln Ala Arg Val Ala Ala

165 170 175

Glu Ala Arg Leu Arg Tyr Leu Ala Asp His Asp Asp Leu Thr Gly Leu

180 185 190

His Asn Arg Arg Ala Leu Leu Gln His Leu Asp Gln Arg Leu Ala Pro

195 200 205

Gly Gln Pro Gly Pro Val Ala Ala Leu Phe Leu Asp Leu Asp Arg Leu

210 215 220

Lys Ala Ile Asn Asp Tyr Leu Gly His Ala Ala Gly Asp Gln Phe Ile

225 230 235 240

His Val Phe Ala Gln Arg Ile Gly Asp Ala Leu Val Gly Glu Ser Leu

245 250 255

Ile Ala Arg Leu Gly Gly Asp Glu Phe Val Leu Ile Pro Ala Ser Pro

260 265 270

Met Ser Ala Asp Ala Ala Gln Pro Leu Ala Glu Arg Leu Arg Asp Gln

275 280 285

Leu Lys Asp His Val Ala Ile Gly Gly Glu Val Leu Thr Arg Thr Val

290 295 300

Ser Ile Gly Val Ala Ser Gly Thr Pro Gly Gln His Thr Pro Ser Asp

305 310 315 320

Leu Leu Arg Arg Ala Asp Gln Ala Ala Leu Ala Ala Lys His Ala Gly

325 330 335

Gly Asp Ser Val Ala Ile Phe Thr Ala Asp Met Ser Val Ser Gly Glu

340 345 350

Leu

<210> 7

<211> 1059

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 7

atgtgcaacg acaccgcgac gccgcagctt gaggagctcg tcaccaccgt agccaaccag 60

ctcatgacag tcgacgctgc cacgtcagcc gaagtcagtc agcgcgtttt ggcctatcta 120

gtggaacagc tgggcgtaga tgtcagcttt ttgcgtcata acgatcgcga caggcgcgcg 180

acgaggctgg tggccgaatg gccacctcgc ctcaacatac cggaccccga tccgctcagg 240

ctgatctact tcgctgatgc cgacccggtg tttgcgctat gcgaacacgc caaagagcct 300

ctcgtgttcc ggcccgagcc ggccaccgag gactatcaac gcctcatcga agaagcccgc 360

ggggttccgg taacgtcggc tgccgccgtg ccgctggtat ctggcgagat caccactgga 420

ctgctggggt tcatcaagtt cggtgatcgg aaatggcacg aggccgagct taacgccctc 480

atgaccatcg ctacactctt cgcccaggtg caggctcgcg tcgccgccga ggcgcggctt 540

cgctatctgg ccgaccatga cgatctgacc ggactgcata accgtcgcgc gttgctgcag 600

cacctggacc aaagactggc ccccggacaa cctggcccgg tcgcggcgct atttctcgac 660

ttggaccgcc tcaaggccat caacgactac ctgggccacg ccgccggtga ccagttcatc 720

catgtgttcg cccaacggat cggtgacgca ctcgttggcg agagcctgat cgcccgactc 780

ggcggcgacg aattcgtcct catacccgca tctccaatga gtgccgatgc cgctcaaccg 840

ctcgccgaac gtcttcgcga ccagctcaag gaccacgtcg ctatcggcgg tgaggtgctc 900

acccgcaccg tcagtatcgg tgtcgcctca gggactcccg gacagcacac accgtcggac 960

ctcctgcgcc gagccgacca agccgctctg gcagccaaac acgccggcgg agatagcgtc 1020

gcgattttca ccgcggacat gtcggtcagc ggcgaactg 1059

<210> 8

<211> 436

<212> PRT

<213> Vibrio cholerae (Vibrio cholerae)

<400> 8

Met Arg Met Thr Trp Asn Phe His Gln Tyr Tyr Thr Asn Arg Asn Asp

1 5 10 15

Gly Leu Met Gly Lys Leu Val Leu Thr Asp Glu Glu Lys Asn Asn Leu

20 25 30

Lys Ala Leu Arg Lys Ile Ile Arg Leu Arg Thr Arg Asp Val Phe Glu

35 40 45

Glu Ala Lys Gly Ile Ala Lys Ala Val Lys Lys Ser Ala Leu Thr Phe

50 55 60

Glu Ile Ile Gln Glu Lys Val Ser Thr Thr Gln Ile Lys His Leu Ser

65 70 75 80

Asp Ser Glu Gln Arg Glu Val Ala Lys Leu Ile Tyr Glu Met Asp Asp

85 90 95

Asp Ala Arg Asp Glu Phe Leu Gly Leu Thr Pro Arg Phe Trp Thr Gln

100 105 110

Gly Ser Phe Gln Tyr Asp Thr Leu Asn Arg Pro Phe Gln Pro Gly Gln

115 120 125

Glu Met Asp Ile Asp Asp Gly Thr Tyr Met Pro Met Pro Ile Phe Glu

130 135 140

Ser Glu Pro Lys Ile Gly His Ser Leu Leu Ile Leu Leu Val Asp Ala

145 150 155 160

Ser Leu Lys Ser Leu Val Ala Glu Asn His Gly Trp Lys Phe Glu Ala

165 170 175

Lys Gln Thr Cys Gly Arg Ile Lys Ile Glu Ala Glu Lys Thr His Ile

180 185 190

Asp Val Pro Met Tyr Ala Ile Pro Lys Asp Glu Phe Gln Lys Lys Gln

195 200 205

Ile Ala Leu Glu Ala Asn Arg Ser Phe Val Lys Gly Ala Ile Phe Glu

210 215 220

Ser Tyr Val Ala Asp Ser Ile Thr Asp Asp Ser Glu Thr Tyr Glu Leu

225 230 235 240

Asp Ser Glu Asn Val Asn Leu Ala Leu Arg Glu Gly Asp Arg Lys Trp

245 250 255

Ile Asn Ser Asp Pro Lys Ile Val Glu Asp Trp Phe Asn Asp Ser Cys

260 265 270

Ile Arg Ile Gly Lys His Leu Arg Lys Val Cys Arg Phe Met Lys Ala

275 280 285

Trp Arg Asp Ala Gln Trp Asp Val Gly Gly Pro Ser Ser Ile Ser Leu

290 295 300

Met Ala Ala Thr Val Asn Ile Leu Asp Ser Val Ala His Asp Ala Ser

305 310 315 320

Asp Leu Gly Glu Thr Met Lys Ile Ile Ala Lys His Leu Pro Ser Glu

325 330 335

Phe Ala Arg Gly Val Glu Ser Pro Asp Ser Thr Asp Glu Lys Pro Leu

340 345 350

Phe Pro Pro Ser Tyr Lys His Gly Pro Arg Glu Met Asp Ile Met Ser

355 360 365

Lys Leu Glu Arg Leu Pro Glu Ile Leu Ser Ser Ala Glu Ser Ala Asp

370 375 380

Ser Lys Ser Glu Ala Leu Lys Lys Ile Asn Met Ala Phe Gly Asn Arg

385 390 395 400

Val Thr Asn Ser Glu Leu Ile Val Leu Ala Lys Ala Leu Pro Ala Phe

405 410 415

Ala Gln Glu Pro Ser Ser Ala Ser Lys Pro Glu Lys Ile Ser Ser Thr

420 425 430

Met Val Ser Gly

435

<210> 9

<211> 1311

<212> DNA

<213> Vibrio cholerae (Vibrio cholerae)

<400> 9

gtgagaatga cttggaactt tcaccagtac tacacaaacc gaaatgatgg cttgatgggc 60

aagctagttc ttacagacga ggagaagaac aatctaaagg cattgcgtaa gatcatccgc 120

ttaagaacac gagatgtatt tgaagaagct aagggtattg ccaaggctgt gaaaaaaagt 180

gctcttacgt ttgaaattat tcaggaaaag gtgtcaacga cccaaattaa gcacctttct 240

gacagcgaac aacgagaagt ggctaagctt atttacgaga tggatgatga tgctcgtgat 300

gagtttttgg gattgacacc tcgcttttgg actcagggaa gctttcagta tgacacgctg 360

aatcgcccgt ttcagcctgg tcaagaaatg gatattgatg atggaaccta tatgccaatg 420

cctatttttg agtcagagcc taagattggt cattctttac taattcttct tgttgacgcg 480

tcacttaagt cacttgtagc tgaaaatcat ggctggaaat ttgaagctaa gcagacttgt 540

gggaggatta agattgaggc agagaaaaca catattgatg taccaatgta tgcaatccct 600

aaagatgagt tccagaaaaa gcaaatagct ttagaagcaa atagatcatt tgttaaaggt 660

gccatttttg aatcatatgt tgcagattca attactgacg atagtgaaac ttatgaatta 720

gattcagaaa acgtaaacct tgctcttcgt gaaggtgatc ggaagtggat caatagcgac 780

cccaaaatag ttgaagattg gttcaacgat agttgtatac gtattggtaa acatcttcgt 840

aaggtttgtc gctttatgaa agcgtggaga gatgcgcagt gggatgttgg aggtccgtca 900

tcgattagtc ttatggctgc aacggtaaat attcttgata gcgttgctca tgatgctagt 960

gatctcggag aaacaatgaa gataattgct aagcatttac ctagtgagtt tgctagggga 1020

gtagagagcc ctgacagtac cgatgaaaag ccactcttcc caccctctta taagcatggc 1080

cctcgggaga tggacattat gagcaaacta gagcgtttgc cagagattct gtcatctgct 1140

gagtcagctg actctaagtc agaggccttg aaaaagatta atatggcgtt tgggaatcgt 1200

gttactaata gcgagcttat tgttttggca aaggctttac cggctttcgc tcaagaacct 1260

agttcagcct cgaaacctga aaaaatcagc agcacaatgg taagtggctg a 1311

<210> 10

<211> 522

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Met Gln Pro Trp His Gly Lys Ala Met Gln Arg Ala Ser Glu Ala Gly

1 5 10 15

Ala Thr Ala Pro Lys Ala Ser Ala Arg Asn Ala Arg Gly Ala Pro Met

20 25 30

Asp Pro Thr Glu Ser Pro Ala Ala Pro Glu Ala Ala Leu Pro Lys Ala

35 40 45

Gly Lys Phe Gly Pro Ala Arg Lys Ser Gly Ser Arg Gln Lys Lys Ser

50 55 60

Ala Pro Asp Thr Gln Glu Arg Pro Pro Val Arg Ala Thr Gly Ala Arg

65 70 75 80

Ala Lys Lys Ala Pro Gln Arg Ala Gln Asp Thr Gln Pro Ser Asp Ala

85 90 95

Thr Ser Ala Pro Gly Ala Glu Gly Leu Glu Pro Pro Ala Ala Arg Glu

100 105 110

Pro Ala Leu Ser Arg Ala Gly Ser Cys Arg Gln Arg Gly Ala Arg Cys

115 120 125

Ser Thr Lys Pro Arg Pro Pro Pro Gly Pro Trp Asp Val Pro Ser Pro

130 135 140

Gly Leu Pro Val Ser Ala Pro Ile Leu Val Arg Arg Asp Ala Ala Pro

145 150 155 160

Gly Ala Ser Lys Leu Arg Ala Val Leu Glu Lys Leu Lys Leu Ser Arg

165 170 175

Asp Asp Ile Ser Thr Ala Ala Gly Met Val Lys Gly Val Val Asp His

180 185 190

Leu Leu Leu Arg Leu Lys Cys Asp Ser Ala Phe Arg Gly Val Gly Leu

195 200 205

Leu Asn Thr Gly Ser Tyr Tyr Glu His Val Lys Ile Ser Ala Pro Asn

210 215 220

Glu Phe Asp Val Met Phe Lys Leu Glu Val Pro Arg Ile Gln Leu Glu

225 230 235 240

Glu Tyr Ser Asn Thr Arg Ala Tyr Tyr Phe Val Lys Phe Lys Arg Asn

245 250 255

Pro Lys Glu Asn Pro Leu Ser Gln Phe Leu Glu Gly Glu Ile Leu Ser

260 265 270

Ala Ser Lys Met Leu Ser Lys Phe Arg Lys Ile Ile Lys Glu Glu Ile

275 280 285

Asn Asp Ile Lys Asp Thr Asp Val Ile Met Lys Arg Lys Arg Gly Gly

290 295 300

Ser Pro Ala Val Thr Leu Leu Ile Ser Glu Lys Ile Ser Val Asp Ile

305 310 315 320

Thr Leu Ala Leu Glu Ser Lys Ser Ser Trp Pro Ala Ser Thr Gln Glu

325 330 335

Gly Leu Arg Ile Gln Asn Trp Leu Ser Ala Lys Val Arg Lys Gln Leu

340 345 350

Arg Leu Lys Pro Phe Tyr Leu Val Pro Lys His Ala Lys Glu Gly Asn

355 360 365

Gly Phe Gln Glu Glu Thr Trp Arg Leu Ser Phe Ser His Ile Glu Lys

370 375 380

Glu Ile Leu Asn Asn His Gly Lys Ser Lys Thr Cys Cys Glu Asn Lys

385 390 395 400

Glu Glu Lys Cys Cys Arg Lys Asp Cys Leu Lys Leu Met Lys Tyr Leu

405 410 415

Leu Glu Gln Leu Lys Glu Arg Phe Lys Asp Lys Lys His Leu Asp Lys

420 425 430

Phe Ser Ser Tyr His Val Lys Thr Ala Phe Phe His Val Cys Thr Gln

435 440 445

Asn Pro Gln Asp Ser Gln Trp Asp Arg Lys Asp Leu Gly Leu Cys Phe

450 455 460

Asp Asn Cys Val Thr Tyr Phe Leu Gln Cys Leu Arg Thr Glu Lys Leu

465 470 475 480

Glu Asn Tyr Phe Ile Pro Glu Phe Asn Leu Phe Ser Ser Asn Leu Ile

485 490 495

Asp Lys Arg Ser Lys Glu Phe Leu Thr Lys Gln Ile Glu Tyr Glu Arg

500 505 510

Asn Asn Glu Phe Pro Val Phe Asp Glu Phe

515 520

<210> 11

<211> 1802

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 11

agcctggggt tccccttcgg gtcgcagact cttgtgtgcc cgccagtagt gcttggtttc 60

caacagctgc tgctggctct tcctcttgcg gccttttcct gaaacggatt cttctttcgg 120

ggaacagaaa gcgccagcca tgcagccttg gcacggaaag gccatgcaga gagcttccga 180

ggccggagcc actgccccca aggcttccgc acggaatgcc aggggcgccc cgatggatcc 240

caccgagtct ccggctgccc ccgaggccgc cctgcctaag gcgggaaagt tcggccccgc 300

caggaagtcg ggatcccggc agaaaaagag cgccccggac acccaggaga ggccgcccgt 360

ccgcgcaact ggggcccgcg ccaaaaaggc ccctcagcgc gcccaggaca cgcagccgtc 420

tgacgccacc agcgcccctg gggcagaggg gctggagcct cctgcggctc gggagccggc 480

tctttccagg gctggttctt gccgccagag gggcgcgcgc tgctccacga agccaagacc 540

tccgcccggg ccctgggacg tgcccagccc cggcctgccg gtctcggccc ccattctcgt 600

acggagggat gcggcgcctg gggcctcgaa gctccgggcg gttttggaga agttgaagct 660

cagccgcgat gatatctcca cggcggcggg gatggtgaaa ggggttgtgg accacctgct 720

gctcagactg aagtgcgact ccgcgttcag aggcgtcggg ctgctgaaca ccgggagcta 780

ctatgagcac gtgaagattt ctgcacctaa tgaatttgat gtcatgttta aactggaagt 840

ccccagaatt caactagaag aatattccaa cactcgtgca tattactttg tgaaatttaa 900

aagaaatccg aaagaaaatc ctctgagtca gtttttagaa ggtgaaatat tatcagcttc 960

taagatgctg tcaaagttta ggaaaatcat taaggaagaa attaacgaca ttaaagatac 1020

agatgtcatc atgaagagga aaagaggagg gagccctgct gtaacacttc ttattagtga 1080

aaaaatatct gtggatataa ccctggcttt ggaatcaaaa agtagctggc ctgctagcac 1140

ccaagaaggc ctgcgcattc aaaactggct ttcagcaaaa gttaggaagc aactacgact 1200

aaagccattt taccttgtac ccaagcatgc aaaggaagga aatggtttcc aagaagaaac 1260

atggcggcta tccttctctc acatcgaaaa ggaaattttg aacaatcatg gaaaatctaa 1320

aacgtgctgt gaaaacaaag aagagaaatg ttgcaggaaa gattgtttaa aactaatgaa 1380

atacctttta gaacagctga aagaaaggtt taaagacaaa aaacatctgg ataaattctc 1440

ttcttatcat gtgaaaactg ccttctttca cgtatgtacc cagaaccctc aagacagtca 1500

gtgggaccgc aaagacctgg gcctctgctt tgataactgc gtgacatact ttcttcagtg 1560

cctcaggaca gaaaaacttg agaattattt tattcctgaa ttcaatctat tctctagcaa 1620

cttaattgac aaaagaagta aggaatttct gacaaagcaa attgaatatg aaagaaacaa 1680

tgagtttcca gtttttgatg aattttgaga ttgtattttt agaaagatct aagaactaga 1740

gtcaccctaa atcctggaga atacaagaaa aatttgaaaa ggggccagac gctgtggctc 1800

ac 1802

<210> 12

<211> 1569

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 12

atgcaaccat ggcacgggaa agccatgcag cgtgcgagcg aagccggggc gacggccccc 60

aaggcgtcgg cgcgtaacgc gcggggtgcg cccatggacc cgacggagtc ccccgcggcg 120

ccggaggcgg ccctgccgaa agcgggtaag ttcggtccag cgcggaaaag cgggagccgc 180

caaaagaagt ccgcgcccga cacccaggag cgtcccccgg tccgggccac cggcgcgcgt 240

gccaaaaaag ccccgcaacg ggcgcaagat acgcagccaa gcgatgcgac ctccgccccc 300

ggggcggagg gtctggagcc cccggccgcc cgggagccag cgctctcgcg cgcgggttcc 360

tgccgtcagc ggggcgcgcg gtgttccacg aaaccccgtc ccccaccagg tccctgggac 420

gtgccgtcgc cgggtttgcc ggtgagcgcg ccaatcctgg tccggcgcga cgcggccccg 480

ggggcgtcga aattgcgtgc ggtgctcgag aaattgaagt tgtcgcgcga cgacatctcc 540

acggccgcgg gtatggtcaa gggcgtggtc gatcatttgt tgttgcggct caagtgtgat 600

tcggcgttcc gcggggtggg cttgctgaac acggggtcct actatgagca tgtcaaaatc 660

agcgccccca acgaatttga cgtgatgttt aagctggaag tgccacgtat ccaattggaa 720

gagtattcca atacccgtgc gtattatttc gtcaaattta agcgcaatcc gaaggaaaat 780

ccactcagcc aattcttgga gggcgaaatt ctgtcggcct cgaaaatgct ctccaaattt 840

cgtaagatta tcaaggagga gatcaacgac attaaggaca cggatgtgat catgaaacgt 900

aaacgtggcg gttcccccgc ggtgacgctc ctcatttcgg aaaaaatttc ggtggacatt 960

accctggcgt tggaatcgaa gtccagctgg ccggcgtcga cccaggaggg cctgcggatt 1020

caaaactggt tgagcgccaa agtgcggaag cagctgcgtc tcaaaccctt ttatttggtc 1080

ccgaaacatg ccaaagaggg taacggtttt caagaggaaa cctggcgttt gagcttctcc 1140

cacattgaga aggagatttt gaacaaccat ggtaagtcca aaacgtgctg cgagaataag 1200

gaagaaaaat gttgtcgcaa agattgtctc aaattgatga aatatttgct ggaacaactc 1260

aaagagcgtt ttaaggacaa gaagcatctc gacaagttct cctcgtatca cgtcaagacc 1320

gccttctttc atgtctgtac gcagaacccg caagatagcc agtgggatcg caaggacttg 1380

gggttgtgtt ttgacaattg cgtcacctat ttcttgcaat gtttgcggac cgagaaattg 1440

gagaactact ttattccaga attcaacttg ttttcctcga atctgattga caaacgctcc 1500

aaagagtttc tgacgaagca gattgaatac gagcgtaaca atgagtttcc ggtctttgac 1560

gagttttga 1569

<210> 13

<211> 10841

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 13

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accaaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc 240

tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc 300

gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 360

tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 420

cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 480

agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 540

aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 600

accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt actatggttg ctttgacgtg 660

cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca 720

ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 780

ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 840

tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccgggg atcctctaga 900

aattccggaa ttgcactcgc cttaggggag tgctaaaaat gatcctggca ctcgcgatca 960

gcgagtgcca ggtcgggacg gtgagaccca gccagcaagc tgtggtcgtc cgtcgcgggc 1020

actgcacccg gccagcgtaa gtaatggggg ttgtcggcac ccggtgacat gcacgctgtg 1080

actcgtccga ccctgcgtga ggctgtcgcc cgcctagccc cgggcactgg gctgcgggac 1140

ggcctggagc gtatcctgcg cggccgcact ggtgccctga tcgtgctggg ccatgacgag 1200

aatgtcgagg ccatctgcga tggtggcttc tccctcgatg tccgctatgc agcaacccgg 1260

ctacgcgagc tgtgcaagat ggacggcgcc gtggtgctgt ccaccgacgg cagccgcatc 1320

gtgcgggcca acgtgcaact ggtaccggat ccgtcgatcc ccaccgacga atcggggacc 1380

cggcaccgct cggccgagcg ggccgcgatc cagaccggtt acccggtgat ctcagtgagc 1440

cactcgatga acatcgtgac cgtctacgtc cgcggggaac gtcacgtatt gaccgactcg 1500

gcaaccatcc tgtcgcgggc caaccaggcc atcgcaaccc tggagcggta caaaaccagg 1560

ctcgacgagg tcagccggca actgtccagg gcagaaatcg aggacttcgt cacgctgcgc 1620

gatgtgatga cggtggtgca acgcctcgag ctggtccggc gaatcgggct ggtgatcgac 1680

tacgacgtgg tcgaactcgg cactgatggt cgtcagctgc ggctgcagct cgacgagttg 1740

ctcggcggca acgacaccgc ccgggaattg atcgtgcgcg attaccacgc caacccggaa 1800

ccaccgtcca cggggcaaat caatgccacc ctggacgaac tggacgccct gtcggacggc 1860

gacctcctcg atttcaccgc gctggcaaag gttttcggat atccgacgac cacggaagcg 1920

caggattcga cgctgagccc gcgtggctac cgcgcgatgg ccggtatccc ccggctccag 1980

ttcgcccatg ccgacctgct ggtccgggcg ttcggaacgt tgcagggtct gctggcggcc 2040

agcgccggcg atctgcaatc agtggacggc atcggcgcca tgtgggcccg tcatgtgcgc 2100

gaggggttgt cacagctggc ggaatcgacc atcagcgatc aataagagca catcgatatg 2160

caaccatggc acgggaaagc catgcagcgt gcgagcgaag ccggggcgac ggcccccaag 2220

gcgtcggcgc gtaacgcgcg gggtgcgccc atggacccga cggagtcccc cgcggcgccg 2280

gaggcggccc tgccgaaagc gggtaagttc ggtccagcgc ggaaaagcgg gagccgccaa 2340

aagaagtccg cgcccgacac ccaggagcgt cccccggtcc gggccaccgg cgcgcgtgcc 2400

aaaaaagccc cgcaacgggc gcaagatacg cagccaagcg atgcgacctc cgcccccggg 2460

gcggagggtc tggagccccc ggccgcccgg gagccagcgc tctcgcgcgc gggttcctgc 2520

cgtcagcggg gcgcgcggtg ttccacgaaa ccccgtcccc caccaggtcc ctgggacgtg 2580

ccgtcgccgg gtttgccggt gagcgcgcca atcctggtcc ggcgcgacgc ggccccgggg 2640

gcgtcgaaat tgcgtgcggt gctcgagaaa ttgaagttgt cgcgcgacga catctccacg 2700

gccgcgggta tggtcaaggg cgtggtcgat catttgttgt tgcggctcaa gtgtgattcg 2760

gcgttccgcg gggtgggctt gctgaacacg gggtcctact atgagcatgt caaaatcagc 2820

gcccccaacg aatttgacgt gatgtttaag ctggaagtgc cacgtatcca attggaagag 2880

tattccaata cccgtgcgta ttatttcgtc aaatttaagc gcaatccgaa ggaaaatcca 2940

ctcagccaat tcttggaggg cgaaattctg tcggcctcga aaatgctctc caaatttcgt 3000

aagattatca aggaggagat caacgacatt aaggacacgg atgtgatcat gaaacgtaaa 3060

cgtggcggtt cccccgcggt gacgctcctc atttcggaaa aaatttcggt ggacattacc 3120

ctggcgttgg aatcgaagtc cagctggccg gcgtcgaccc aggagggcct gcggattcaa 3180

aactggttga gcgccaaagt gcggaagcag ctgcgtctca aaccctttta tttggtcccg 3240

aaacatgcca aagagggtaa cggttttcaa gaggaaacct ggcgtttgag cttctcccac 3300

attgagaagg agattttgaa caaccatggt aagtccaaaa cgtgctgcga gaataaggaa 3360

gaaaaatgtt gtcgcaaaga ttgtctcaaa ttgatgaaat atttgctgga acaactcaaa 3420

gagcgtttta aggacaagaa gcatctcgac aagttctcct cgtatcacgt caagaccgcc 3480

ttctttcatg tctgtacgca gaacccgcaa gatagccagt gggatcgcaa ggacttgggg 3540

ttgtgttttg acaattgcgt cacctatttc ttgcaatgtt tgcggaccga gaaattggag 3600

aactacttta ttccagaatt caacttgttt tcctcgaatc tgattgacaa acgctccaaa 3660

gagtttctga cgaagcagat tgaatacgag cgtaacaatg agtttccggt ctttgacgag 3720

ttttgaaagc ttgagatggt gagcaagggc gaggaggata acatggccat catcaaggag 3780

ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag 3840

ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag 3900

ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag 3960

gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc 4020

ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac 4080

tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc 4140

tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg 4200

taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc 4260

ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc 4320

ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc 4380

gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac 4440

aagtagacta gttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact 4500

cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga 4560

actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc 4620

tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac 4680

gttgcgaagc aacggcccgg aagggtggcg ggcaggacgc ccgccataaa ctgccaggca 4740

tcaaattaag cagaaggcca tcctgacgga tggccttttt tctagagtcg accaccaagg 4800

gcaccatctc tgcttgggcc accccgttgg ccgcagccag ctcgctgaga gccgtgaacg 4860

acagggcgaa cgccagcccg ccgacggcga gggttccgac cgctgcaact cccggtgcaa 4920

ccttgtcccg gtctattctc ttcactgcac cagctccaat ctggtgtgaa tgcccctcgt 4980

ctgttcgcgc aggcgggggg ctctattcgt ttgtcagcat cgaaagtagc cagatcaggg 5040

atgcgttgca accgcgtatg cccaggtcag aagagtcgca caagagttgc agacccctgg 5100

aaagaaaaat ggccagaggg cgaaaacacc ctctgaccag cggagcgggc gacgggaatc 5160

gaacccgcgt agctagtttg gaagaatggg tgtctgccga ccacatatgg gccggtcaag 5220

ataggttttt accccctctc ggctgcatcc tctaagtgga aagaaattgc aggtcgtaga 5280

agcgcgttga agcctgagag ttgcacagga gttgcaaccc ggtagccttg ttcacgacga 5340

gaggagacct agttggcacg tcgcggatgg ggatcgctga agactcagcg cagcgggagg 5400

atccaagcct catacgtcaa cccgcaggac ggtgtgaggt actacgcgct gcagacctac 5460

gacaacaaga tggacgccga agcctggctc gcgggcgaga agcggctcat cgagatggag 5520

acctggaccc ctccacagga ccgggcgaag aaggcagccg ccagcgccat cacgctggag 5580

gagtacaccc ggaagtggct cgtggagcgc gacctcgcag acggcaccag ggatctgtac 5640

agcgggcacg cggagcgccg catctacccg gtgctaggtg aagtggcggt cacagagatg 5700

acgccagctc tggtgcgtgc gtggtgggcc gggatgggta ggaagcaccc gactgcccgc 5760

cggcatgcct acaacgtcct ccgggcggtg atgaacacag cggtcgagga caagctgatc 5820

gcagagaacc cgtgccggat cgagcagaag gcagccgatg agcgcgacgt agaggcgctg 5880

acgcctgagg agctggacat cgtcgccgct gagatcttcg agcactaccg gatcgcggca 5940

tacatcctgg cgtggacgag cctccggttc ggagagctga tcgagcttcg ccgcaaggac 6000

atcgtggacg acggcatgac gatgaagctc cgggtgcgcc gtggcgcttc ccgcgtgggg 6060

aacaagatcg tcgttggcaa cgccaagacc gtccggtcga agcgtcctgt gacggttccg 6120

cctcacgtcg cggagatgat ccgagcgcac atgaaggacc gtacgaagat gaacaagggc 6180

cccgaggcat tcctggtgac cacgacgcag ggcaaccggc tgtcgaagtc cgcgttcacc 6240

aagtcgctga agcgtggcta cgccaagatc ggtcggccgg aactccgcat ccacgacctc 6300

cgcgctgtcg gcgctacgtt cgccgctcag gcaggtgcga cgaccaagga gctgatggcc 6360

cgtctcggtc acacgactcc taggatggcg atgaagtacc agatggcgtc tgaggcccgc 6420

gacgaggcta tcgctgaggc gatgtccaag ctggccaaga cctcctgaaa cgcaaaaagc 6480

ccccctccca aggacactga gtcctaaaga ggggggtttc ttgtcagtac gcgaagaacc 6540

acgcctggcc gcgagcgcca gcaccgccgc tctgtgcgga gacctgggca ccagccccgc 6600

cgccgccagg agcattgccg ttcccgccag ctgagttctg ttgtgcgccg cctatgtaga 6660

gctggtcgtt gtaggtccga tctccaggcg actttccggc gacgctgagg atgtcgatca 6720

cagagcctcc gggaccgccg gttgcggtca aacctgacca tccgacagcg gacgccgtgg 6780

tgtttcctcc agggcctccg gccttgcctg agaatacaga gccagctccc gctgcgcctc 6840

cagctccgac gagcccggtg atcgtcttgg tcgacctgca ggcatgcaaa agctgatcct 6900

tgccgagctg ggatggaagc ccggccgacc caccctggag gagatgatcg aggatgccag 6960

ggcctttcac gcccgccgct gctgagcgtc cgccgccggg cccgcaccgc cgtcggccgg 7020

cccgctccgg gctcgcagca gcgggcttcg gcgcgggccc ggggctcccg ggccgccggg 7080

cggggctccg cccggcggcc gccgggggcc gggggcggcg ccgggcggcc cggggcgtca 7140

ggcgccgggg gcggtgtccg gcggccccca gaggaactgc gccagttcct ccggatcggt 7200

gaagccggag agatccagcg gggtctcctc gaacacctcg aagtcgtgca ggaaggtgaa 7260

ggcgagcagt tcgcgggcga agtcctcggt ccgcttccac tgcgccccgt cgagcagcgc 7320

ggccaggatc tcgcggtcgc cccggaaggc gttgagatgc agttgcacca ggctgtagcg 7380

ggagtctccc gcatagacgt cggtgaagtc gacgatcccg gtgacctcgg tcgcggccag 7440

gtccacgaag atgttggtcc cgtgcaggtc gccgtggacg aaccggggtt cgcggccggc 7500

cagcagcgtg tccacgtccg gcagccagtc ctccaggcgg tccagcagcc ggggcgagag 7560

gtagccccac ccgcggtggt cctcgacggt cgccgcgcgg cgttcccgca gcagttccgg 7620

gaagacctcg gaatgggggg tgagcacggt gttcccggtc agcggcaccc tgtgcagccg 7680

gccgagcacc cggccgagtt cgcgggccag ggcgagcagc gcgttccggt cggtcgtgcc 7740

gtccatcgcg gaccgccagg tggtgccggt catccggctc atcaccaggt agggccacgg 7800

ccaggctccg gtgccgggcc gcagctcgcc gcggccgagg aggcggggca ccggcaccgg 7860

ggcgtccgcc aggaccgcgt acgcctccga ctccgacgcg aggctctccg gaccgcacca 7920

gtgctcgccg aacagcttga tcaccgggcc gggctcgccg accagtacgg ggttggtgct 7980

ctcgccgggc acccgcagca ccggcggcac cggcagcccg agctcctcca gggctcggcg 8040

ggccagcggc tcccagaatt cctggtcgtt ccgcaggctc gcgtaggaat catccgaatc 8100

aatacggtcg agaagtaaca gggattcttg tgtcacagcg gacctctatt cacagggtac 8160

gggccggctt aattccgcac ggccggtcgc gacacggcct gtccgcaccg cggatcaggc 8220

gttgacgatg acgggctggt cggccacgtc ggggacgttc tcggtggtgc tgcggtcggg 8280

atcgccaatc tctacgggcc gaccgaggcg acggtgtacg ccaccgcctg gttctgcgac 8340

ggcgaggcgc cgtcccaggc cccgccgatc cccgtccccc gcgtcgtcga gcgcggtgcc 8400

gacgacaccg ccgcgtggct cgtcacggag gccgtccccg gcgtcgcggc ggccgaggag 8460

tggcccgagc accagcggtt cgccgtggtc gaggcgatgg cggagctggc ccgcgccctc 8520

cacgagctgc ccgtggagga ctgccccttc gaccggcgcc tcgacgcggc ggtcgccgag 8580

gcccggcgga acgtcgccga gggcctgtgg acctcgacga cctgcaggca tgcaagctag 8640

cttttgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 8700

ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 8760

ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 8820

cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 8880

tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 8940

aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 9000

aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 9060

tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 9120

ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 9180

cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag 9240

ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 9300

ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 9360

gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 9420

agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 9480

cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 9540

aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 9600

aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 9660

ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 9720

aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 9780

ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 9840

agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 9900

cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 9960

ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 10020

gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 10080

cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 10140

cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 10200

ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 10260

catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 10320

tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 10380

ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 10440

catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 10500

cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 10560

cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 10620

acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 10680

ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 10740

tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 10800

attaacctat aaaaataggc gtatcacgag gccctttcgt c 10841

<210> 14

<211> 316

<212> PRT

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 14

Met Asp Ala Val Gly Ala Ala Ala Leu Leu Ser Ala Ala Ala Arg Val

1 5 10 15

Gly Val Val Cys His Val His Pro Asp Ala Asp Thr Ile Gly Ala Gly

20 25 30

Leu Ala Leu Ala Leu Val Leu Asp Gly Cys Gly Lys Arg Val Glu Val

35 40 45

Ser Phe Ala Ala Pro Ala Thr Leu Pro Glu Ser Leu Arg Ser Leu Pro

50 55 60

Gly Cys His Leu Leu Val Arg Pro Glu Val Met Arg Arg Asp Val Asp

65 70 75 80

Leu Val Val Thr Val Asp Ile Pro Ser Val Asp Arg Leu Gly Ala Leu

85 90 95

Gly Asp Leu Thr Asp Ser Gly Arg Glu Leu Leu Val Ile Asp His His

100 105 110

Ala Ser Asn Asp Leu Phe Gly Thr Ala Asn Phe Ile Asp Pro Ser Ala

115 120 125

Asp Ser Thr Thr Thr Met Val Ala Glu Ile Leu Asp Ala Trp Gly Lys

130 135 140

Pro Ile Asp Pro Arg Val Ala His Cys Ile Tyr Ala Gly Leu Ala Thr

145 150 155 160

Asp Thr Gly Ser Phe Arg Trp Ala Ser Val Arg Gly Tyr Arg Leu Ala

165 170 175

Ala Arg Leu Val Glu Ile Gly Val Asp Asn Ala Thr Val Ser Arg Thr

180 185 190

Leu Met Asp Ser His Pro Phe Thr Trp Leu Pro Leu Leu Ser Arg Val

195 200 205

Leu Gly Ser Ala Gln Leu Val Ser Glu Ala Val Gly Gly Arg Gly Leu

210 215 220

Val Tyr Val Val Val Asp Asn Arg Glu Trp Val Ala Ala Arg Ser Glu

225 230 235 240

Glu Val Glu Ser Ile Val Asp Ile Val Arg Thr Thr Gln Gln Ala Glu

245 250 255

Val Ala Ala Val Phe Lys Glu Val Glu Pro His Arg Trp Ser Val Ser

260 265 270

Met Arg Ala Lys Thr Val Asn Leu Ala Ala Val Ala Ser Gly Phe Gly

275 280 285

Gly Gly Gly His Arg Leu Ala Ala Gly Tyr Thr Thr Thr Gly Ser Ile

290 295 300

Asp Asp Ala Val Ala Ser Leu Arg Ala Ala Leu Gly

305 310 315

<210> 15

<211> 951

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 15

gtggacgccg tcggtgccgc tgcgctgttg tcggccgctg ccagggtcgg ggtagtctgc 60

cacgtccacc ccgatgccga caccatcggc gccggattgg cattggcatt ggtgttggac 120

gggtgcggca agcgggtaga ggtcagcttt gccgcgccgg cgacactgcc cgagtcgctg 180

cgttcgctgc cgggctgcca tctgctggtc cgccctgagg tgatgcgccg cgatgtcgat 240

ttggttgtga ctgttgacat tccgagtgtt gatcggctcg gtgctctggg cgatctaact 300

gattccgggc gggagctcct ggtaatcgac catcacgcct ccaacgacct gttcggcacc 360

gcgaatttca ttgacccgtc ggcggattcc accacgacga tggttgccga gatcctcgac 420

gcgtggggga aaccgataga cccgcgcgtc gcgcactgca tctacgccgg gttggcgacc 480

gacacggggt cgtttcgctg ggccagtgtg cgggggtatc ggctggcggc gcggctggta 540

gagatcggtg tggacaacgc caccgtcagc aggaccttga tggacagcca tcccttcacc 600

tggttgccgt tgctatcgcg ggtgttgggt tcggcgcagc tggtgtccga ggcggtcggt 660

ggccgcgggc tggtttacgt cgtcgtcgac aaccgggagt gggtcgctgc gcgctcggag 720

gaagtggaaa gcatcgtcga catcgtccgc accacgcaac aagccgaggt cgcggcggtg 780

ttcaaggagg tcgaaccgca tcggtggtcg gtgtcgatgc gggctaagac cgtgaatttg 840

gccgcggttg cctctgggtt cggtggcggt ggtcaccggc tggccgcggg gtatacgacc 900

accggctcga tcgacgacgc tgtggcgtcg ttgcgcgcgg cgcttggtta g 951

<210> 16

<211> 336

<212> PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400> 16

Met Thr Thr Ile Asp Pro Arg Ser Glu Leu Val Asp Gly Arg Arg Arg

1 5 10 15

Ala Gly Ala Arg Val Asp Ala Val Gly Ala Ala Ala Leu Leu Ser Ala

20 25 30

Ala Ala Arg Val Gly Val Val Cys His Val His Pro Asp Ala Asp Thr

35 40 45

Ile Gly Ala Gly Leu Ala Leu Ala Leu Val Leu Asp Gly Cys Gly Lys

50 55 60

Arg Val Glu Val Ser Phe Ala Ala Pro Ala Thr Leu Pro Glu Ser Leu

65 70 75 80

Arg Ser Leu Pro Gly Cys His Leu Leu Val Arg Pro Glu Val Met Arg

85 90 95

Arg Asp Val Asp Leu Val Val Thr Val Asp Ile Pro Ser Val Asp Arg

100 105 110

Leu Gly Ala Leu Gly Asp Leu Thr Asp Ser Gly Arg Glu Leu Leu Val

115 120 125

Ile Asp His His Ala Ser Asn Asp Leu Phe Gly Thr Ala Asn Phe Ile

130 135 140

Asp Pro Ser Ala Asp Ser Thr Thr Thr Met Val Ala Glu Ile Leu Asp

145 150 155 160

Ala Trp Gly Lys Pro Ile Asp Pro Arg Val Ala His Cys Ile Tyr Ala

165 170 175

Gly Leu Ala Thr Asp Thr Gly Ser Phe Arg Trp Ala Ser Val Arg Gly

180 185 190

Tyr Arg Leu Ala Ala Arg Leu Val Glu Ile Gly Val Asp Asn Ala Thr

195 200 205

Val Ser Arg Thr Leu Met Asp Ser His Pro Phe Thr Trp Leu Pro Leu

210 215 220

Leu Ser Arg Val Leu Gly Ser Ala Gln Leu Val Ser Glu Ala Val Gly

225 230 235 240

Gly Arg Gly Leu Val Tyr Val Val Val Asp Asn Arg Glu Trp Val Ala

245 250 255

Ala Arg Ser Glu Glu Val Glu Ser Ile Val Asp Ile Val Arg Thr Thr

260 265 270

Gln Gln Ala Glu Val Ala Ala Val Phe Lys Glu Val Glu Pro His Arg

275 280 285

Trp Ser Val Ser Met Arg Ala Lys Thr Val Asn Leu Ala Ala Val Ala

290 295 300

Ser Gly Phe Gly Gly Gly Gly His Arg Leu Ala Ala Gly Tyr Thr Thr

305 310 315 320

Thr Gly Ser Ile Asp Asp Ala Val Ala Ser Leu Arg Ala Ala Leu Gly

325 330 335

<210> 17

<211> 288

<212> PRT

<213> Unknown (Unknown)

<220>

<223> description unknown: diguanylate phosphodiesterase sequences

<400> 17

Met Ile Asp Tyr Glu Glu Met Phe Arg Gly Ala Met Gln Ala Arg Ala

1 5 10 15

Met Val Ala Asn Pro Asp Gln Trp Ala Asp Ser Asp Arg Asp Gln Val

20 25 30

Asn Thr Arg His Tyr Leu Ser Thr Ser Met Arg Val Ala Leu Asp Arg

35 40 45

Gly Glu Phe Phe Leu Val Tyr Gln Pro Ile Ile Arg Leu Ala Asp Asn

50 55 60

Arg Ile Ile Gly Ala Glu Ala Leu Leu Arg Trp Glu His Pro Thr Leu

65 70 75 80

Gly Thr Leu Leu Pro Gly Arg Phe Ile Asp Arg Ala Glu Asn Asn Gly

85 90 95

Leu Met Val Pro Leu Thr Ala Phe Val Leu Glu Gln Ala Cys Arg His

100 105 110

Val Arg Ser Trp Arg Asp His Ser Thr Asp Pro Gln Pro Phe Val Ser

115 120 125

Val Asn Val Ser Ala Ser Thr Ile Cys Asp Pro Gly Phe Leu Val Leu

130 135 140

Val Glu Gly Val Leu Gly Glu Thr Gly Leu Pro Ala His Ala Leu Gln

145 150 155 160

Leu Glu Leu Ala Glu Asp Ala Arg Leu Ser Arg Asp Glu Lys Ala Val

165 170 175

Thr Arg Leu Gln Glu Leu Ser Ala Leu Gly Val Gly Ile Ala Ile Asp

180 185 190

Asp Phe Gly Ile Gly Phe Ser Ser Leu Ala Tyr Leu Pro Arg Leu Pro

195 200 205

Val Asp Val Val Lys Leu Gly Gly Lys Phe Ile Glu Cys Leu Asp Gly

210 215 220

Asp Ile Gln Ala Arg Leu Ala Asn Glu Gln Ile Thr Arg Ala Met Ile

225 230 235 240

Asp Leu Gly Asp Lys Leu Gly Ile Thr Val Thr Ala Lys Leu Val Glu

245 250 255

Ser Pro Ser Gln Ala Ala Arg Leu Arg Ala Phe Gly Cys Lys Ala Ala

260 265 270

Gln Gly Trp His Phe Ala Lys Ala Leu Pro Val Asp Phe Phe Arg Glu

275 280 285

<210> 18

<211> 867

<212> DNA

<213> Unknown (Unknown)

<220>

<223> description unknown: diguanylate phosphodiesterase sequences

<400> 18

ttgatcgact acgaagagat gtttaggggc gcgatgcaag cgcgagcgat ggtagccaat 60

cctgaccaat gggcggactc cgaccgcgac caggtcaaca ctcgccatta tctgtccact 120

tcgatgcgcg tggcactgga tcgcggtgaa ttcttcctcg tctaccagcc aatcatccgg 180

cttgccgaca accgcatcat cggcgccgag gccctgctgc gctgggaaca cccgacgttg 240

ggcacgctac tcccgggccg gttcatcgac cgtgccgaga acaacggact gatggtgccg 300

ctcacggcct tcgtgctcga gcaggcctgc cgccacgtcc gcagttggcg tgaccacagc 360

accgacccgc aaccgtttgt cagcgtcaac gtctccgcca gcaccatctg cgatcccggc 420

ttcctggtgc tggtcgaagg tgtgctcggc gaaaccggcc tgcccgccca tgccctgcag 480

ctcgaactgg ccgaggacgc gcgccttagc agagacgaga aggcggtgac caggctacaa 540

gaattgtccg ctctcggcgt cggcatcgcc atcgacgact tcggcattgg attctccagc 600

ctcgcctacc ttccccgcct ccccgtcgac gtggtcaaac tcgggggaaa gttcatcgag 660

tgcctcgatg gcgacattca agctcggctg gccaacgaac agatcacccg ggcaatgatc 720

gaccttggcg acaagctcgg tatcaccgtc actgcaaagc tagtcgaaag ccccagccaa 780

gccgcccggt tgcgcgcctt cggctgtaaa gccgcacaag gctggcactt tgccaaggca 840

ctgccggtcg actttttcag agagtag 867

<210> 19

<211> 307

<212> PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400> 19

Met Asp Arg Cys Cys Gln Arg Ala Thr Ala Phe Ala Cys Ala Leu Arg

1 5 10 15

Pro Thr Lys Leu Ile Asp Tyr Glu Glu Met Phe Arg Gly Ala Met Gln

20 25 30

Ala Arg Ala Met Val Ala Asn Pro Asp Gln Trp Ala Asp Ser Asp Arg

35 40 45

Asp Gln Val Asn Thr Arg His Tyr Leu Ser Thr Ser Met Arg Val Ala

50 55 60

Leu Asp Arg Gly Glu Phe Phe Leu Val Tyr Gln Pro Ile Ile Arg Leu

65 70 75 80

Ala Asp Asn Arg Ile Ile Gly Ala Glu Ala Leu Leu Arg Trp Glu His

85 90 95

Pro Thr Leu Gly Thr Leu Leu Pro Gly Arg Phe Ile Asp Arg Ala Glu

100 105 110

Asn Asn Gly Leu Met Val Pro Leu Thr Ala Phe Val Leu Glu Gln Ala

115 120 125

Cys Arg His Val Arg Ser Trp Arg Asp His Ser Thr Asp Pro Gln Pro

130 135 140

Phe Val Ser Val Asn Val Ser Ala Ser Thr Ile Cys Asp Pro Gly Phe

145 150 155 160

Leu Val Leu Val Glu Gly Val Leu Gly Glu Thr Gly Leu Pro Ala His

165 170 175

Ala Leu Gln Leu Glu Leu Ala Glu Asp Ala Arg Leu Ser Arg Asp Glu

180 185 190

Lys Ala Val Thr Arg Leu Gln Glu Leu Ser Ala Leu Gly Val Gly Ile

195 200 205

Ala Ile Asp Asp Phe Gly Ile Gly Phe Ser Ser Leu Ala Tyr Leu Pro

210 215 220

Arg Leu Pro Val Asp Val Val Lys Leu Gly Gly Lys Phe Ile Glu Cys

225 230 235 240

Leu Asp Gly Asp Ile Gln Ala Arg Leu Ala Asn Glu Gln Ile Thr Arg

245 250 255

Ala Met Ile Asp Leu Gly Asp Lys Leu Gly Ile Thr Val Thr Ala Lys

260 265 270

Leu Val Glu Thr Pro Ser Gln Ala Ala Arg Leu Arg Ala Phe Gly Cys

275 280 285

Lys Ala Ala Gln Gly Trp His Phe Ala Lys Ala Leu Pro Val Asp Phe

290 295 300

Phe Arg Glu

305

<210> 20

<211> 1350

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 20

atgacgattc ctgcgttcca tcccggtgaa ctcaatgtgt actcggcacc gggggatgtc 60

gccgatgtca gtcgcgcact gcgactcacc ggccggcgag tgatgttggt gcctactatg 120

ggtgcgctgc acgaaggcca cctcgcgttg gtgcgtgcgg ccaagcgggt gcccggatcg 180

gtcgtcgtcg tgtcgatctt cgtcaacccg atgcaattcg gtgccgggga agatctcgac 240

gcctatcccc gcaccccgga cgacgacctg gcgcaactgc gggccgaagg cgtggaaatc 300

gctttcacgc caactaccgc ggcgatgtat cccgacggcc tgcgcaccac cgtgcaaccc 360

ggtccgttgg ccgccgaact cgagggcggc ccgcggccaa cccatttcgc cggcgtgctg 420

acggtcgtgc taaagctgct gcagatcgtg cgcccggatc gggtgttctt cggtgagaag 480

gactaccagc agctggtgct gatccggcag ctggtcgcgg acttcaacct cgatgtcgcg 540

gtggtcggcg tgccgaccgt gcgcgaagcc gacgggctgg cgatgtcgtc gcgcaaccgc 600

tacctggacc cggcccagcg tgcggcggcc gtcgcgctct cggcggcgct aacggccgca 660

gcgcatgcgg caacggctgg cgcgcaggcc gcgctggatg ccgcccgtgc ggtgctcgac 720

gctgcacccg gcgtggcggt cgactacctg gagctgcgcg atatcgggct tggcccgatg 780

ccgctcaacg gttccggtcg gctgctggtt gctgcccggc ttggcaccac caggctgctg 840

gacaacattg cgattgaaat cggaactttc gccggcaccg accgcccgga cggataccgg 900

gcaatcctcg aatcacattg gagaaactga tgttacggac gatgctgaag tcgaagatcc 960

accgcgccac ggtgacctgc gccgacctgc actacgtcgg ctcggtgacc atcgatgccg 1020

acttgatgga cgccgccgac ctgctggaag gcgaacaggt aaccatcgtc gatatcgaca 1080

acggtgctcg actggtcacc tacgcgatca ccggcgaacg cggcagtggt gtgattggca 1140

tcaacggtgc cgccgcgcac ttggtgcatc cgggggatct ggtgattctg attgcgtacg 1200

cgacgatgga cgacgcccgg gcccgcacat accagccgcg gatcgtgttt gtcgacgctt 1260

acaacaaacc gatcgacatg ggccacgatc cggcatttgt gcccgaaaac gcgggcgagc 1320

tgctagaccc ccggctcggt gtgggatagc 1350

<210> 21

<211> 309

<212> PRT

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 21

Met Thr Ile Pro Ala Phe His Pro Gly Glu Leu Asn Val Tyr Ser Ala

1 5 10 15

Pro Gly Asp Val Ala Asp Val Ser Arg Ala Leu Arg Leu Thr Gly Arg

20 25 30

Arg Val Met Leu Val Pro Thr Met Gly Ala Leu His Glu Gly His Leu

35 40 45

Ala Leu Val Arg Ala Ala Lys Arg Val Pro Gly Ser Val Val Val Val

50 55 60

Ser Ile Phe Val Asn Pro Met Gln Phe Gly Ala Gly Glu Asp Leu Asp

65 70 75 80

Ala Tyr Pro Arg Thr Pro Asp Asp Asp Leu Ala Gln Leu Arg Ala Glu

85 90 95

Gly Val Glu Ile Ala Phe Thr Pro Thr Thr Ala Ala Met Tyr Pro Asp

100 105 110

Gly Leu Arg Thr Thr Val Gln Pro Gly Pro Leu Ala Ala Glu Leu Glu

115 120 125

Gly Gly Pro Arg Pro Thr His Phe Ala Gly Val Leu Thr Val Val Leu

130 135 140

Lys Leu Leu Gln Ile Val Arg Pro Asp Arg Val Phe Phe Gly Glu Lys

145 150 155 160

Asp Tyr Gln Gln Leu Val Leu Ile Arg Gln Leu Val Ala Asp Phe Asn

165 170 175

Leu Asp Val Ala Val Val Gly Val Pro Thr Val Arg Glu Ala Asp Gly

180 185 190

Leu Ala Met Ser Ser Arg Asn Arg Tyr Leu Asp Pro Ala Gln Arg Ala

195 200 205

Ala Ala Val Ala Leu Ser Ala Ala Leu Thr Ala Ala Ala His Ala Ala

210 215 220

Thr Ala Gly Ala Gln Ala Ala Leu Asp Ala Ala Arg Ala Val Leu Asp

225 230 235 240

Ala Ala Pro Gly Val Ala Val Asp Tyr Leu Glu Leu Arg Asp Ile Gly

245 250 255

Leu Gly Pro Met Pro Leu Asn Gly Ser Gly Arg Leu Leu Val Ala Ala

260 265 270

Arg Leu Gly Thr Thr Arg Leu Leu Asp Asn Ile Ala Ile Glu Ile Gly

275 280 285

Thr Phe Ala Gly Thr Asp Arg Pro Asp Gly Tyr Arg Ala Ile Leu Glu

290 295 300

Ser His Trp Arg Asn

305

<210> 22

<211> 139

<212> PRT

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 22

Met Leu Arg Thr Met Leu Lys Ser Lys Ile His Arg Ala Thr Val Thr

1 5 10 15

Cys Ala Asp Leu His Tyr Val Gly Ser Val Thr Ile Asp Ala Asp Leu

20 25 30

Met Asp Ala Ala Asp Leu Leu Glu Gly Glu Gln Val Thr Ile Val Asp

35 40 45

Ile Asp Asn Gly Ala Arg Leu Val Thr Tyr Ala Ile Thr Gly Glu Arg

50 55 60

Gly Ser Gly Val Ile Gly Ile Asn Gly Ala Ala Ala His Leu Val His

65 70 75 80

Pro Gly Asp Leu Val Ile Leu Ile Ala Tyr Ala Thr Met Asp Asp Ala

85 90 95

Arg Ala Arg Thr Tyr Gln Pro Arg Ile Val Phe Val Asp Ala Tyr Asn

100 105 110

Lys Pro Ile Asp Met Gly His Asp Pro Ala Phe Val Pro Glu Asn Ala

115 120 125

Gly Glu Leu Leu Asp Pro Arg Leu Gly Val Gly

130 135

<210> 23

<211> 2501

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 23

atgacgattc ctgcgttcca tcccggtgaa ctcaatgtgt actcggcacc gggggatgtc 60

gccgatgtca gtcgcgcact gcgactcacc ggccggcgag tgatgttggt gcctactatg 120

ggtgcgctgc acgaaggcca cctcgcgttg gtgcgtgcgg ccaagcgggt gcccggatcg 180

gtcgtcgtcg tgtcgatctt cgtcaacccg atgcaattcg gtgccgggga agatctcgac 240

gcctatcccc gcaccccgga cgacgacctg gcgcaactgc gggccgaagg cgtggaaatc 300

gctttcacgc caactaccgc ggcgatgtat cccgacggcc tgcgcaccac cgtgcaaccc 360

ggtccgttgg ccgccgaact cgagggcggc ccgcggccaa cccatttcgc cggcgtgctg 420

acggtcgtgc taaagctgct gcagatcgtg cgcccggatc gggtgttctt cggtgagaag 480

gactaccagc agctggtgct gatccggcag ctggtcgcgg acttcaacct cgatgtcgcg 540

gtggtcggcg tgccgaccgt gcgcgaagcc gacgggctgg cgatgtcgtc gcgcaaccgc 600

tacctggacc cggcccagcg tgcggcggcc gtcgcgctct cggcggcgct aacggccgca 660

gcgcatgcgg caacggctgg cgcgcaggcc gcgctggatg ccgcccgtgc ggtgctcgac 720

gctgcacccg gcgtggcgtg gcggtcgact acctggagct gcgcgatatc gggcttggcc 780

cgatgccgct caacggttcc ggtcggctgc tggttgctgc ccggcttggc accaccaggc 840

tgctggacaa cattgcgatt gaaatcggaa ctttcgccgg caccgaccgc ccggacggat 900

accgggcaat cctcgaatca cattggagaa actgatgtta cggacgatgc tgaagtcgaa 960

gatccaccgc gccacggtga cctgcgccga cctgcactac gtcggctcgg tgaccatcga 1020

tgccgacttg atggacgccg ccgacctgct ggaaggcgaa caggtaacca tcgtcgatat 1080

cgacaacggt gctcgactgg tcacctacgc gatcaccggc gaacgcggca gtggtgtgat 1140

tggcatcaac ggtgccgccg cgcacttggt gcatccgggg gatctggtga ttctgattgc 1200

gtacgcgacg atggacgacg cccgggcccg cacataccag ccgcggatcg tgtttgtcga 1260

cgcttacaac aaaccgatcg acatgggcca cgatccggca tttgtgcccg aaaacgcggg 1320

cgagctgcta gacccccggc tcggtgtggg atagccgtgc tgctggcgat tgacgtccgc 1380

aacacccaca ccgttgtggg cctgctgtcc ggaatgaaag agcacgcaaa ggtcgtgcag 1440

cagtggcgga tacgcaccga atccgaagtc accgccgacg aactggcact gacgatcgac 1500

gggctgatcg gcgaggattc cgagcggctc accggtaccg ccgccttgtc cacggtcccg 1560

tccgtgctgc acgaggtgcg gataatgctc gaccagtact ggccgtcggt gccgcacgtg 1620

ctgatcgagc ccggagtacg caccgggatc cctttgctcg tcgacaaccc gaaggaagtg 1680

ggcgcagacc gcatcgtgaa ctgtttggcc gcctatgacc ggttccggaa ggccgccatc 1740

gtcgttgact ttggatcctc gatctgtgtt gatgttgtat cggccaaggg tgaatttctt 1800

ggcggcgcca tcgcgcccgg ggtgcaggtg tcttccgatg ccgcggcggc ccgctcggcg 1860

gcattgcgcc gcgttgaact tgcccgccca cgttcggtgg ttggcaagaa caccgtcgaa 1920

tgcatgcaag ccggtgcggt gttcggcttc gccgggctgg tagacgggtt ggtaggccgc 1980

atccgcgagg acgtgtccgg tttctccgtc gaccacgatg tcgcgatcgt ggctaccggg 2040

cataccgcgc ccctgctgct gccggaattg cacaccgtcg accattacga ccagcacctg 2100

accttgcagg gtctgcggct ggtgttcgag cgtaacctcg aagtccagcg cggccggctc 2160

aagacggcgc gctgacgtcg atgccggcat cgagtctggg taccgggtcg cccgccgccg 2220

acaggctcga cgccacccac gagcgtcggc gtgaggtcat ttaagctggc acgtcgtgag 2280

tgccgctgac acagcagaag accttcctga gcagttccgg attcgccggg acaagcgcgc 2340

tcgcttgctg gcccaggggc gcgatcccta tcccgtcgcg gtgccgcgca ctcacacgtt 2400

ggccgaggtt cgcgccgccc accctgactt gccgatcgat accgcgaccg aagacatcgt 2460

cggcgtcgcg ggccgagtga tctttgcgcg caactcggga a 2501

<210> 24

<211> 724

<212> PRT

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 24

Met Thr Ile Pro Ala Phe His Pro Gly Glu Leu Asn Val Tyr Ser Ala

1 5 10 15

Pro Gly Asp Val Ala Asp Val Ser Arg Ala Leu Arg Leu Thr Gly Arg

20 25 30

Arg Val Met Leu Val Pro Thr Met Gly Ala Leu His Glu Gly His Leu

35 40 45

Ala Leu Val Arg Ala Ala Lys Arg Val Pro Gly Ser Val Val Val Val

50 55 60

Ser Ile Phe Val Asn Pro Met Gln Phe Gly Ala Gly Glu Asp Leu Asp

65 70 75 80

Ala Tyr Pro Arg Thr Pro Asp Asp Asp Leu Ala Gln Leu Arg Ala Glu

85 90 95

Gly Val Glu Ile Ala Phe Thr Pro Thr Thr Ala Ala Met Tyr Pro Asp

100 105 110

Gly Leu Arg Thr Thr Val Gln Pro Gly Pro Leu Ala Ala Glu Leu Glu

115 120 125

Gly Gly Pro Arg Pro Thr His Phe Ala Gly Val Leu Thr Val Val Leu

130 135 140

Lys Leu Leu Gln Ile Val Arg Pro Asp Arg Val Phe Phe Gly Glu Lys

145 150 155 160

Asp Tyr Gln Gln Leu Val Leu Ile Arg Gln Leu Val Ala Asp Phe Asn

165 170 175

Leu Asp Val Ala Val Val Gly Val Pro Thr Val Arg Glu Ala Asp Gly

180 185 190

Leu Ala Met Ser Ser Arg Asn Arg Tyr Leu Asp Pro Ala Gln Arg Ala

195 200 205

Ala Ala Val Ala Leu Ser Ala Ala Leu Thr Ala Ala Ala His Ala Ala

210 215 220

Thr Ala Gly Ala Gln Ala Ala Leu Asp Ala Ala Arg Ala Val Leu Asp

225 230 235 240

Ala Ala Pro Gly Val Ala Trp Arg Ser Thr Thr Trp Ser Cys Ala Ile

245 250 255

Ser Gly Leu Ala Arg Cys Arg Ser Thr Val Pro Val Gly Cys Trp Leu

260 265 270

Leu Pro Gly Leu Ala Pro Pro Gly Cys Trp Thr Thr Leu Arg Leu Lys

275 280 285

Ser Glu Leu Ser Pro Ala Pro Thr Ala Arg Thr Asp Thr Gly Gln Ser

290 295 300

Ser Asn His Ile Gly Glu Thr Asp Val Thr Asp Asp Ala Glu Val Glu

305 310 315 320

Asp Pro Pro Arg His Gly Asp Leu Arg Arg Pro Ala Leu Arg Arg Leu

325 330 335

Gly Asp His Arg Cys Arg Leu Asp Gly Arg Arg Arg Pro Ala Gly Arg

340 345 350

Arg Thr Gly Asn His Arg Arg Tyr Arg Gln Arg Cys Ser Thr Gly His

355 360 365

Leu Arg Asp His Arg Arg Thr Arg Gln Trp Cys Asp Trp His Gln Arg

370 375 380

Cys Arg Arg Ala Leu Gly Ala Ser Gly Gly Ser Gly Asp Ser Asp Cys

385 390 395 400

Val Arg Asp Asp Gly Arg Arg Pro Gly Pro His Ile Pro Ala Ala Asp

405 410 415

Arg Val Cys Arg Arg Leu Gln Gln Thr Asp Arg His Gly Pro Arg Ser

420 425 430

Gly Ile Cys Ala Arg Lys Arg Gly Arg Ala Ala Arg Pro Pro Ala Arg

435 440 445

Cys Gly Ile Ala Val Leu Leu Ala Ile Asp Val Arg Asn Thr His Thr

450 455 460

Val Val Gly Leu Leu Ser Gly Met Lys Glu His Ala Lys Val Val Gln

465 470 475 480

Gln Trp Arg Ile Arg Thr Glu Ser Glu Val Thr Ala Asp Glu Leu Ala

485 490 495

Leu Thr Ile Asp Gly Leu Ile Gly Glu Asp Ser Glu Arg Leu Thr Gly

500 505 510

Thr Ala Ala Leu Ser Thr Val Pro Ser Val Leu His Glu Val Arg Ile

515 520 525

Met Leu Asp Gln Tyr Trp Pro Ser Val Pro His Val Leu Ile Glu Pro

530 535 540

Gly Val Arg Thr Gly Ile Pro Leu Leu Val Asp Asn Pro Lys Glu Val

545 550 555 560

Gly Ala Asp Arg Ile Val Asn Cys Leu Ala Ala Tyr Asp Arg Phe Arg

565 570 575

Lys Ala Ala Ile Val Val Asp Phe Gly Ser Ser Ile Cys Val Asp Val

580 585 590

Val Ser Ala Lys Gly Glu Phe Leu Gly Gly Ala Ile Ala Pro Gly Val

595 600 605

Gln Val Ser Ser Asp Ala Ala Ala Ala Arg Ser Ala Ala Leu Arg Arg

610 615 620

Val Glu Leu Ala Arg Pro Arg Ser Val Val Gly Lys Asn Thr Val Glu

625 630 635 640

Cys Met Gln Ala Gly Ala Val Phe Gly Phe Ala Gly Leu Val Asp Gly

645 650 655

Leu Val Gly Arg Ile Arg Glu Asp Val Ser Gly Phe Ser Val Asp His

660 665 670

Asp Val Ala Ile Val Ala Thr Gly His Thr Ala Pro Leu Leu Leu Pro

675 680 685

Glu Leu His Thr Val Asp His Tyr Asp Gln His Leu Thr Leu Gln Gly

690 695 700

Leu Arg Leu Val Phe Glu Arg Asn Leu Glu Val Gln Arg Gly Arg Leu

705 710 715 720

Lys Thr Ala Arg

<210> 25

<211> 139

<212> PRT

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 25

Met Leu Arg Thr Met Leu Lys Ser Lys Ile His Arg Ala Thr Val Thr

1 5 10 15

Cys Ala Asp Leu His Tyr Val Gly Ser Val Thr Ile Asp Ala Asp Leu

20 25 30

Met Asp Ala Ala Asp Leu Leu Glu Gly Glu Gln Val Thr Ile Val Asp

35 40 45

Ile Asp Asn Gly Ala Arg Leu Val Thr Tyr Ala Ile Thr Gly Glu Arg

50 55 60

Gly Ser Gly Val Ile Gly Ile Asn Gly Ala Ala Ala His Leu Val His

65 70 75 80

Pro Gly Asp Leu Val Ile Leu Ile Ala Tyr Ala Thr Met Asp Asp Ala

85 90 95

Arg Ala Arg Thr Tyr Gln Pro Arg Ile Val Phe Val Asp Ala Tyr Asn

100 105 110

Lys Pro Ile Asp Met Gly His Asp Pro Ala Phe Val Pro Glu Asn Ala

115 120 125

Gly Glu Leu Leu Asp Pro Arg Leu Gly Val Gly

130 135

<210> 26

<211> 930

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 26

atgacgattc ctgcgttcca tcccggtgaa ctcaatgtgt actcggcacc gggggatgtc 60

gccgatgtca gtcgcgcact gcgactcacc ggccggcgag tgatgttggt gcctactatg 120

ggtgcgctgc acgaaggcca cctcgcgttg gtgcgtgcgg ccaagcgggt gcccggatcg 180

gtcgtcgtcg tgtcgatctt cgtcaacccg atgcaattcg gtgccgggga agatctcgac 240

gcctatcccc gcaccccgga cgacgacctg gcgcaactgc gggccgaagg cgtggaaatc 300

gctttcacgc caactaccgc ggcgatgtat cccgacggcc tgcgcaccac cgtgcaaccc 360

ggtccgttgg ccgccgaact cgagggcggc ccgcggccaa cccatttcgc cggcgtgctg 420

acggtcgtgc taaagctgct gcagatcgtg cgcccggatc gggtgttctt cggtgagaag 480

gactaccagc agctggtgct gatccggcag ctggtcgcgg acttcaacct cgatgtcgcg 540

gtggtcggcg tgccgaccgt gcgcgaagcc gacgggctgg cgatgtcgtc gcgcaaccgc 600

tacctggacc cggcccagcg tgcggcggcc gtcgcgctct cggcggcgct aacggccgca 660

gcgcatgcgg caacggctgg cgcgcaggcc gcgctggatg ccgcccgtgc ggtgctcgac 720

gctgcacccg gcgtggcggt cgactacctg gagctgcgcg atatcgggct tggcccgatg 780

ccgctcaacg gttccggtcg gctgctggtt gctgcccggc ttggcaccac caggctgctg 840

gacaacattg cgattgaaat cggaactttc gccggcaccg accgcccgga cggataccgg 900

gcaatcctcg aatcacattg gagaaactga 930

<210> 27

<211> 420

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 27

atgttacgga cgatgctgaa gtcgaagatc caccgcgcca cggtgacctg cgccgacctg 60

cactacgtcg gctcggtgac catcgatgcc gacttgatgg acgccgccga cctgctggaa 120

ggcgaacagg taaccatcgt cgatatcgac aacggtgctc gactggtcac ctacgcgatc 180

accggcgaac gcggcagtgg tgtgattggc atcaacggtg ccgccgcgca cttggtgcat 240

ccgggggatc tggtgattct gattgcgtac gcgacgatgg acgacgcccg ggcccgcaca 300

taccagccgc ggatcgtgtt tgtcgacgct tacaacaaac cgatcgacat gggccacgat 360

ccggcatttg tgcccgaaaa cgcgggcgag ctgctagacc cccggctcgg tgtgggatag 420

<210> 28

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 28

ggggatgacg attcctgcg 19

<210> 29

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 29

gggctatccc acaccgagc 19

<210> 30

<211> 7738

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 30

ggatccttct agaattccgg aattgcactc gccttagggg agtgctaaaa atgatcctgg 60

cactcgcgat cagcgagtgc caggtcggga cggtgagacc cagccagcaa gctgtggtcg 120

tccgtcgcgg gcactgcacc cggccagcgt aagtaatggg ggttgtcggc acccggtgac 180

ctagacacat gcatgcatgc ttaattaatt aagcgatatc cggaggaatc acttccatat 240

gcacgctgtg actcgtccga ccctgcgtga ggctgtcgcc cgcctagccc cgggcactgg 300

gctgcgggac ggcctggagc gtatcctgcg cggccgcact ggtgccctga tcgtgctggg 360

ccatgacgag aatgtcgagg ccatctgcga tggtggcttc tccctcgatg tccgctatgc 420

agcaacccgg ctacgcgagc tgtgcaagat ggacggcgcc gtggtgctgt ccaccgacgg 480

cagccgcatc gtgcgggcca acgtgcaact ggtaccggat ccgtcgatcc ccaccgacga 540

atcggggacc cggcaccgct cggccgagcg ggccgcgatc cagaccggtt acccggtgat 600

ctcagtgagc cactcgatga acatcgtgac cgtctacgtc cgcggggaac gtcacgtatt 660

gaccgactcg gcaaccatcc tgtcgcgggc caaccaggcc atcgcaaccc tggagcggta 720

caaaaccagg ctcgacgagg tcagccggca actgtccagg gcagaaatcg aggacttcgt 780

cacgctgcgc gatgtgatga cggtggtgca acgcctcgag ctggtccggc gaatcgggct 840

ggtgatcgac tacgacgtgg tcgaactcgg cactgatggt cgtcagctgc ggctgcagct 900

cgacgagttg ctcggcggca acgacaccgc ccgggaattg atcgtgcgcg attaccacgc 960

caacccggaa ccaccgtcca cggggcaaat caatgccacc ctggacgaac tggacgccct 1020

gtcggacggc gacctcctcg atttcaccgc gctggcaaag gttttcggat atccgacgac 1080

cacggaagcg caggattcga cgctgagccc gcgtggctac cgcgcgatgg ccggtatccc 1140

ccggctccag ttcgcccatg ccgacctgct ggtccgggcg ttcggaacgt tgcagggtct 1200

gctggcggcc agcgccggcg atctgcaatc agtggacggc atcggcgcca tgtgggcccg 1260

tcatgtgcgc gaggggttgt cacagctggc ggaatcgacc atcagcgatc aataacgcgt 1320

tctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 1380

atggcgaatg gcgctttgcc tggtttccgg tcgaagcttg gccggatcta aagttttgtc 1440

gtctttccag acgttagtaa atgaattttc tgtatgaggt tttgctaaac aactttcaac 1500

agtttcagcg gagtgagaat agaaaggaac aactaaagga attgcgaata ataatttttt 1560

cacgttgaaa atctccaaaa aaaaaggctc caaaaggagc ctttaattgt atcggtttat 1620

cagcttgctt tcgaggtgaa tttcttaaac agcttgatac cgatagttgc gccgacaatg 1680

acaacaacca tcgcccacgc ataaccgata tattcggtcg ctgaggcttg cagggagtca 1740

aaggccgctt ttgcggggat ccgctcggag gcgcggtcgc ggcgcggctg tggcatgtcg 1800

gggcgtgccg ctcccccggc gccgcccatc ggcccgccca ttggcattcc gcccatgccg 1860

cccatcattc ctgtggagcc agaactgatc cagcctgtgc cacagccgac aggatggtga 1920

ccaccatttg ccccatatca ccgtcggtac tgatcccgtc gtcaataaac cgaaccgcta 1980

caccctgagc atcaaactct tttatcagtt ggatcatgtc ggcggtgtcg cggccaagac 2040

ggtcgagctt cttcaccaga atgacatcac cttcctccac cttcatcctc agcaaatcca 2100

gcccttcccg atctgttgaa ctgccggatg ccttgtcggt aaagatgcgg ttagctttta 2160

cccctgcatc tttgagcgct gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca 2220

ggcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt 2280

gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt 2340

gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag 2400

ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt aaccaattct 2460

gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 2520

ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc 2580

cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 2640

cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 2700

actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc 2760

cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat 2820

tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc 2880

gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga 2940

tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca 3000

tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag 3060

tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga 3120

aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg 3180

acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc 3240

ggcctcgagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt 3300

atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat 3360

cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag 3420

gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa 3480

atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg 3540

ctggatgatg gggcgattca ggcctggtat gagtcagcaa caccttcttc acgaggcaga 3600

cctcagcgct agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg 3660

aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata 3720

caggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg 3780

agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa 3840

cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag 3900

catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac 3960

caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc 4020

ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg 4080

gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg 4140

cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact 4200

ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag 4260

gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca 4320

aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt 4380

cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt attaaggggt 4440

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4500

ggatcttcac ctagatcctt ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 4560

aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 4620

actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4680

aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4740

tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4800

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4860

ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 4920

ggccctttcg tcttcaagaa ttcccaggca tcaaataaaa cgaaaggctc agtcgaaaga 4980

ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 5040

gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 5100

gccataaact gccagggaat tcccatcgag ccgagaacgt tatcgaagtt ggtcatgtgt 5160

aatcccctcg tttgaacttt ggattaagcg tagatacacc cttggacaag ccagttggat 5220

tcggagacaa gcaaattcag ccttaaaaag ggcgaggccc tgcggtggtg gaacaccgca 5280

gggcctctaa ccgctcgacg cgctgcacca accagcccgc gaacggctgg cagccagcgt 5340

aaggcgcggc tcatcgggcg gcgttcgcca cgatgtcctg cacttcgagc caagcctcga 5400

acacctgctg gtgtgcacga ctcacccggt tgttgacacc gcgcgcggcc gtgcgggctc 5460

ggtggggcgg ctctgtcgcc cttgccagcg tgagtagcgc gtacctcacc tcgcccaaca 5520

ggtcgcacac agccgattcg tacgccataa agccaggtga gcccaccagc tccgtaagtt 5580

cgggcgctgt gtggctcgta cccgcgcatt caggcggcag ggggtctaac gggtctaagg 5640

cggcgtgtac gcggccacag cggctctcag cggcccggaa acgtcctcga aacgacgcat 5700

gtgttcctcc tggttggtac aggtggttgg gggtgctcgg ctgtcgcggt tgttccacca 5760

ccagggctcg acgggagagc gggggagtgt gcagttgtgg ggtggcccct cagcgaaata 5820

tctgacttgg agctcgtgtc ggaccataca ccggtgatta atcgtggtct actaccaagc 5880

gtgagccacg tcgccgacga atttgagcag ctctggctgc cgtactggcc gctggcaagc 5940

gacgatctgc tcgaggggat ctaccgccaa agccgcgcgt cggccctagg ccgccggtac 6000

atcgaggcga acccaacagc gctggcaaac ctgctggtcg tggacgtaga ccatccagac 6060

gcagcgctcc gagcgctcag cgcccggggg tcccatccgc tgcccaacgc gatcgtgggc 6120

aatcgcgcca acggccacgc acacgcagtg tgggcactca acgcccctgt tccacgcacc 6180

gaatacgcgc ggcgtaagcc gctcgcatac atggcggcgt gcgccgaagg ccttcggcgg 6240

ccgtcgacgg cgaccgcagt tactcaggcc tcatgaccaa aaaccccggc cacatcgcct 6300

gggaaacgga atggctccac tcagatctct acacactcag ccacatcgag gccgagctcg 6360

gcgcgaacat gccaccgccg cgctggcgtc agcagaccac gtacaaagcg gctccgacgc 6420

cgctagggcg gaattgcgca ctgttcgatt ccgtcaggtt gtgggcctat cgtcccgccc 6480

tcatgcggat ctacctgccg acccggaacg tggacggact cggccgcgcg atctatgccg 6540

agtgccacgc gcgaaacgcc gaattcccgt gcaacgacgt gtgtcccgga ccgctaccgg 6600

acagcgaggt ccgcgccatc gccaacagca tttggcgttg gatcacaacc aagtcgcgca 6660

tttgggcgga cgggatcgtg gtctacgagg ccacactcag tgcgcgccag tcggccatct 6720

cgcggaaggg cgcagcagcg cgcacggcgg cgagcacagt tgcgcggcgc gcaaagtccg 6780

cgtcagccat ggaggcattg ctatgagcga cggctacagc gacggctaca gcgacggcta 6840

caaccggcag ccgactgtcc gcaaaaagcc gtgacgcgcc gaaggcgctc gaatcaccgg 6900

actatccgaa cgccacgtcg tccggctcgt ggcgcaggaa cgcagcgagt ggctcgccga 6960

gcaggctgca cgcgcgcgaa gcatccgcgc ctatcacgac gacgagggcc actcttggcc 7020

gcaaacggcc aaacatttcg ggctgcatct ggacaccgtt aagcgactcg gctatcgggc 7080

gaggaaagag cgtgcggcag aacaggaagc ggctcaaaag gcccacaacg aagccgacaa 7140

tccaccgctg ttctaacgca attggggacg ggtgtcgcgg gggttccgtg gggggttccg 7200

ttgcaacggg tcggacaggt aaaagtcctg gtagacgcta gttttctggt ttgggccatg 7260

cctgtctcgt tgcgtgtttc gttgcgccgt tttgaatacc agccagacga gacggggttc 7320

tacgaatctt ggtcgatacc aagccatttc cgctgaatat cggggagctc accgccagaa 7380

tcggtggttg tggtgatgta cgtggcgaac tccgttgtag tgcctgtggt ggcatccgtg 7440

gccactctcg ttgcacggtt cgttgtgccg ttacaggccc cgttgacagc tcaccgaacg 7500

tagttaaaac atgctggtca aactaggttt accaacgata cgagtcagct catctagggc 7560

cagttctagg cgttgttcgt tgcgcggttc gttgcgcatg tttcgtgtgg ttgctagatg 7620

gctccgcaac cacacgcttc gaggttgagt gcttccagca cgggcgcgat ccagaagaac 7680

ttcgtcgtgc gactgtcctc gttgggatct agcccgccta atgagcgggc tttttttt 7738

<210> 31

<211> 8534

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 31

cttctagaat tccggaattg cactcgcctt aggggagtgc taaaaatgat cctggcactc 60

gcgatcagcg agtgccaggt cgggacggtg agacccagcc agcaagctgt ggtcgtccgt 120

cgcgggcact gcacccggcc agcgtaagta atgggggttg tcggcacccg gtgacctaga 180

cacatgcatg catgcttaat taattaagcg atatccggag gaatcacttc catatgcacg 240

ctgtgactcg tccgaccctg cgtgaggctg tcgcccgcct agccccgggc actgggctgc 300

gggacggcct ggagcgtatc ctgcgcggcc gcactggtgc cctgatcgtg ctgggccatg 360

acgagaatgt cgaggccatc tgcgatggtg gcttctccct cgatgtccgc tatgcagcaa 420

cccggctacg cgagctgtgc aagatggacg gcgccgtggt gctgtccacc gacggcagcc 480

gcatcgtgcg ggccaacgtg caactggtac cggatccgtc gatccccacc gacgaatcgg 540

ggacccggca ccgctcggcc gagcgggccg cgatccagac cggttacccg gtgatctcag 600

tgagccactc gatgaacatc gtgaccgtct acgtccgcgg ggaacgtcac gtattgaccg 660

actcggcaac catcctgtcg cgggccaacc aggccatcgc aaccctggag cggtacaaaa 720

ccaggctcga cgaggtcagc cggcaactgt ccagggcaga aatcgaggac ttcgtcacgc 780

tgcgcgatgt gatgacggtg gtgcaacgcc tcgagctggt ccggcgaatc gggctggtga 840

tcgactacga cgtggtcgaa ctcggcactg atggtcgtca gctgcggctg cagctcgacg 900

agttgctcgg cggcaacgac accgcccggg aattgatcgt gcgcgattac cacgccaacc 960

cggaaccacc gtccacgggg caaatcaatg ccaccctgga cgaactggac gccctgtcgg 1020

acggcgacct cctcgatttc accgcgctgg caaaggtttt cggatatccg acgaccacgg 1080

aagcgcagga ttcgacgctg agcccgcgtg gctaccgcgc gatggccggt atcccccggc 1140

tccagttcgc ccatgccgac ctgctggtcc gggcgttcgg aacgttgcag ggtctgctgg 1200

cggccagcgc cggcgatctg caatcagtgg acggcatcgg cgccatgtgg gcccgtcatg 1260

tgcgcgaggg gttgtcacag ctggcggaat cgaccatcag cgatcaataa cgcgttctgg 1320

cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 1380

gaatggcgct ttgcctggtt tccggtcgaa gcttggccgg atctaaagtt ttgtcgtctt 1440

tccagacgtt agtaaatgaa ttttctgtat gaggttttgc taaacaactt tcaacagttt 1500

cagcggagtg agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcacgt 1560

tgaaaatctc caaaaaaaaa ggctccaaaa ggagccttta attgtatcgg tttatcagct 1620

tgctttcgag gtgaatttct taaacagctt gataccgata gttgcgccga caatgacaac 1680

aaccatcgcc cacgcataac cgatatattc ggtcgctgag gcttgcaggg agtcaaaggc 1740

cgcttttgcg gggatccgct cggaggcgcg gtcgcggcgc ggctgtggca tgtcggggcg 1800

tgccgctccc ccggcgccgc ccatcggccc gcccattggc attccgccca tgccgcccat 1860

cattcctgtg gagccagaac tgatccagcc tgtgccacag ccgacaggat ggtgaccacc 1920

atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 1980

tgagcatcaa actcttttat cagttggatc atgtcggcgg tgtcgcggcc aagacggtcg 2040

agcttcttca ccagaatgac atcaccttcc tccaccttca tcctcagcaa atccagccct 2100

tcccgatctg ttgaactgcc ggatgccttg tcggtaaaga tgcggttagc ttttacccct 2160

gcatctttga gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca taccaggcct 2220

gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt 2280

aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg 2340

gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc 2400

gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatga 2460

tcagctatcc cacaccgagc cgggggtcta gcagctcgcc cgcgttttcg ggcacaaatg 2520

ccggatcgtg gcccatgtcg atcggtttgt tgtaagcgtc gacaaacacg atccgcggct 2580

ggtatgtgcg ggcccgggcg tcgtccatcg tcgcgtacgc aatcagaatc accagatccc 2640

ccggatgcac caagtgcgcg gcggcaccgt tgatgccaat cacaccactg ccgcgttcgc 2700

cggtgatcgc gtaggtgacc agtcgagcac cgttgtcgat atcgacgatg gttacctgtt 2760

cgccttccag caggtcggcg gcgtccatca agtcggcatc gatggtcacc gagccgacgt 2820

agtgcaggtc ggcgcaggtc accgtggcgc ggtggatctt cgacttcagc atcgtccgta 2880

acatcagttt ctccaatgtg attcgaggat tgcccggtat ccgtccgggc ggtcggtgcc 2940

ggcgaaagtt ccgatttcaa tcgcaatgtt gtccagcagc ctggtggtgc caagccgggc 3000

agcaaccagc agccgaccgg aaccgttgag cggcatcggg ccaagcccga tatcgcgcag 3060

ctccaggtag tcgaccgcca cgccgggtgc agcgtcgagc accgcacggg cggcatccag 3120

cgcggcctgc gcgccagccg ttgccgcatg cgctgcggcc gttagcgccg ccgagagcgc 3180

gacggccgcc gcacgctggg ccgggtccag gtagcggttg cgcgacgaca tcgccagccc 3240

gtcggcttcg cgcacggtcg gcacgccgac caccgcgaca tcgaggttga agtccgcgac 3300

cagctgccgg atcagcacca gctgctggta gtccttctca ccgaagaaca cccgatccgg 3360

gcgcacgatc tgcagcagct ttagcacgac cgtcagcacg ccggcgaaat gggttggccg 3420

cgggccgccc tcgagttcgg cggccaacgg accgggttgc acggtggtgc gcaggccgtc 3480

gggatacatc gccgcggtag ttggcgtgaa agcgatttcc acgccttcgg cccgcagttg 3540

cgccaggtcg tcgtccgggg tgcggggata ggcgtcgaga tcttccccgg caccgaattg 3600

catcgggttg acgaagatcg acacgacgac gaccgatccg ggcacccgct tggccgcacg 3660

caccaacgcg aggtggcctt cgtgcagcgc acccatagta ggcaccaaca tcactcgccg 3720

gccggtgagt cgcagtgcgc gactgacatc ggcgacatcc cccggtgccg agtacacatt 3780

gagttcaccg ggatggaacg caggaatcgt catgccgtca aaacctcgac gacatccgcg 3840

ggggcgtgtg cgcgctgcgc ggtccgcagc gcgtttatcc ggtatgcctg ggccagcgct 3900

gcgtcgacgt ccgcgagggc cgccagatga tccgcgaccg ctgccgcatc gccgcgggcg 3960

accggtccgg tgagcgcggc ctgtccccgc tgcagcgtgt tctccagcgc cgctctggcc 4020

agcggcccga cgatgcgctc cacgatcccg cccggctggt cgtactagta acaccccttg 4080

tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg 4140

caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg 4200

ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc gtggcaaagc 4260

aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac cgtggctccc 4320

tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca acaccttctt 4380

cacgaggcag acctcagcgc tagcggagtg tatactggct tactatgttg gcactgatga 4440

gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag 4500

aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt 4560

cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg 4620

aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc 4680

cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac 4740

tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct 4800

ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca 4860

ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag 4920

tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca 4980

aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg 5040

cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt 5100

acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt 5160

tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct 5220

tattaagggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5280

attatcaaaa aggatcttca cctagatcct tttaaaagtg ctcatcattg gaaaacgttc 5340

ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 5400

tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 5460

aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 5520

catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 5580

atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 5640

aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 5700

gcgtatcacg aggccctttc gtcttcaaga attcccaggc atcaaataaa acgaaaggct 5760

cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 5820

aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg 5880

gcaggacgcc cgccataaac tgccagggaa ttcccatcga gccgagaacg ttatcgaagt 5940

tggtcatgtg taatcccctc gtttgaactt tggattaagc gtagatacac ccttggacaa 6000

gccagttgga ttcggagaca agcaaattca gccttaaaaa gggcgaggcc ctgcggtggt 6060

ggaacaccgc agggcctcta accgctcgac gcgctgcacc aaccagcccg cgaacggctg 6120

gcagccagcg taaggcgcgg ctcatcgggc ggcgttcgcc acgatgtcct gcacttcgag 6180

ccaagcctcg aacacctgct ggtgtgcacg actcacccgg ttgttgacac cgcgcgcggc 6240

cgtgcgggct cggtggggcg gctctgtcgc ccttgccagc gtgagtagcg cgtacctcac 6300

ctcgcccaac aggtcgcaca cagccgattc gtacgccata aagccaggtg agcccaccag 6360

ctccgtaagt tcgggcgctg tgtggctcgt acccgcgcat tcaggcggca gggggtctaa 6420

cgggtctaag gcggcgtgta cgcggccaca gcggctctca gcggcccgga aacgtcctcg 6480

aaacgacgca tgtgttcctc ctggttggta caggtggttg ggggtgctcg gctgtcgcgg 6540

ttgttccacc accagggctc gacgggagag cgggggagtg tgcagttgtg gggtggcccc 6600

tcagcgaaat atctgacttg gagctcgtgt cggaccatac accggtgatt aatcgtggtc 6660

tactaccaag cgtgagccac gtcgccgacg aatttgagca gctctggctg ccgtactggc 6720

cgctggcaag cgacgatctg ctcgagggga tctaccgcca aagccgcgcg tcggccctag 6780

gccgccggta catcgaggcg aacccaacag cgctggcaaa cctgctggtc gtggacgtag 6840

accatccaga cgcagcgctc cgagcgctca gcgcccgggg gtcccatccg ctgcccaacg 6900

cgatcgtggg caatcgcgcc aacggccacg cacacgcagt gtgggcactc aacgcccctg 6960

ttccacgcac cgaatacgcg cggcgtaagc cgctcgcata catggcggcg tgcgccgaag 7020

gccttcggcg gccgtcgacg gcgaccgcag ttactcaggc ctcatgacca aaaaccccgg 7080

ccacatcgcc tgggaaacgg aatggctcca ctcagatctc tacacactca gccacatcga 7140

ggccgagctc ggcgcgaaca tgccaccgcc gcgctggcgt cagcagacca cgtacaaagc 7200

ggctccgacg ccgctagggc ggaattgcgc actgttcgat tccgtcaggt tgtgggccta 7260

tcgtcccgcc ctcatgcgga tctacctgcc gacccggaac gtggacggac tcggccgcgc 7320

gatctatgcc gagtgccacg cgcgaaacgc cgaattcccg tgcaacgacg tgtgtcccgg 7380

accgctaccg gacagcgagg tccgcgccat cgccaacagc atttggcgtt ggatcacaac 7440

caagtcgcgc atttgggcgg acgggatcgt ggtctacgag gccacactca gtgcgcgcca 7500

gtcggccatc tcgcggaagg gcgcagcagc gcgcacggcg gcgagcacag ttgcgcggcg 7560

cgcaaagtcc gcgtcagcca tggaggcatt gctatgagcg acggctacag cgacggctac 7620

agcgacggct acaaccggca gccgactgtc cgcaaaaagc cgtgacgcgc cgaaggcgct 7680

cgaatcaccg gactatccga acgccacgtc gtccggctcg tggcgcagga acgcagcgag 7740

tggctcgccg agcaggctgc acgcgcgcga agcatccgcg cctatcacga cgacgagggc 7800

cactcttggc cgcaaacggc caaacatttc gggctgcatc tggacaccgt taagcgactc 7860

ggctatcggg cgaggaaaga gcgtgcggca gaacaggaag cggctcaaaa ggcccacaac 7920

gaagccgaca atccaccgct gttctaacgc aattggggac gggtgtcgcg ggggttccgt 7980

ggggggttcc gttgcaacgg gtcggacagg taaaagtcct ggtagacgct agttttctgg 8040

tttgggccat gcctgtctcg ttgcgtgttt cgttgcgccg ttttgaatac cagccagacg 8100

agacggggtt ctacgaatct tggtcgatac caagccattt ccgctgaata tcggggagct 8160

caccgccaga atcggtggtt gtggtgatgt acgtggcgaa ctccgttgta gtgcctgtgg 8220

tggcatccgt ggccactctc gttgcacggt tcgttgtgcc gttacaggcc ccgttgacag 8280

ctcaccgaac gtagttaaaa catgctggtc aaactaggtt taccaacgat acgagtcagc 8340

tcatctaggg ccagttctag gcgttgttcg ttgcgcggtt cgttgcgcat gtttcgtgtg 8400

gttgctagat ggctccgcaa ccacacgctt cgaggttgag tgcttccagc acgggcgcga 8460

tccagaagaa cttcgtcgtg cgactgtcct cgttgggatc tagcccgcct aatgagcggg 8520

cttttttttg gatc 8534

<210> 32

<211> 8801

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<220>

<221> modified_base

<222> (5756)..(5756)

<223> a, c, t, g, unknown or otherwise

<400> 32

cggaccgcta ccggacagcg aggtccgcgc catcgccaac agcatttggc gttggatcac 60

aaccaagtcg cgcatttggg cggacgggat cgtggtctac gaggccacac tcagtgcgcg 120

ccagtcggcc atctcgcgga agggcgcagc agcgcgcacg gcggcgagca cagttgcgcg 180

gcgcgcaaag tccgcgtcag ccatggaggc attgctatga gcgacggcta cagcgacggc 240

tacaaccggc agccgactgt ccgcaaaaag cggcgcgtga ccgccgccga aggcgctcga 300

atcaccggac tatccgaacg ccacgtcgtc cggctcgtgg cgcaggaacg cagcgagtgg 360

ctcgccgagc aggctgcacg ccgcgaacgc atccgcgcct atcacgacga cgagggccac 420

tcttggccgc aaacggccaa acatttcggg ctgcatctgg acaccgttaa gcgactcggc 480

tatcgggcga ggaaagagcg tgcggcagaa caggaagcgg ctcaaaaggc ccacaacgaa 540

gccgacaatc caccgctgtt ctaacgcaat tggggagcgg gtgtcgcggg ggttccgtgg 600

ggggttccgt tgcaacgggt cggacaggta aaagtcctgg tagacgctag ttttctggtt 660

tgggccatgc ctgtctcgtt gcgtgtttcg ttgcgcccgt tttgaatacc agccagacga 720

gacggggttc tacgaatctt ggtcgatacc aagccatttc cgctgaatat cggggagctc 780

accgccagaa tcggtggttg tggtgatgta cgtggcgaac tccgttgtag tgcctgtggt 840

ggcatccgtg gcgcggccgc ggtaccagat ctttaaatct agataaagaa gtgacgcggt 900

ctcaagcgtc gagcgtcgcc agcgtgtcga ggatgtcgaa gtcgtagccg tcggcgctgg 960

cgatgtagac ctgctggtcg aattgactgt cgcgcataca catcgggccc cggggcccgt 1020

cgaacccgac atcgtgcgcg gatgccatca ggtccggtat ctcgggggag tgggcccgct 1080

ggaagatggc ctcgagcgca agcagaccct cgtaacagga ttcggccatc gcgttgagcg 1140

gtggcgcgtc ggcgccgtag cgggcgacgt agctgcccat caggtccatg gcacccgcgg 1200

tggccagtga actgaagtac gccgcggcga catagaggtt ttcggtggag ccggcgccgc 1260

tggccagcag catgttctcc tccatcagcg ggctgaaccg cgccatgcgg tcgtgcccgc 1320

cggcgcgcgc gaactcgcgg ttgaacaaca cggcgtcctg gccgacgagc agcatcaaca 1380

cggcctgcgc ccccgacgcg atggccttgc ggacaggtgc gcggaaatcg tcggtgccgt 1440

acgggacgta gatctcccgt ctgagctcga ggtccagatc tcggcagtac gcgcgggcgg 1500

ccgcggcgga acggcgcggc cagatgtagt catcgccgac caggcaccag gaccggatgc 1560

cgaagtggtc gcgcagccag gcgagcgcgg gcgcgatctg gatctgcggt gtctcgcctg 1620

tgcagaacac gcccggtgtg cgttcaccgc cctcgtacaa cgaggtgtag acgtacggga 1680

tgcggtcgcg gaccaccggg gagatgcggt tgcgcacggc cgagatgtgc cagccggtca 1740

cggcgtcgag accgtgacct cgcaaccggt cggcgacggt ccgggcgacg tcgtcgccgg 1800

gcgctccgcc gtcgagcacc tcgatggtga ccttgcggcc ctgcaggccg cctcggtcgt 1860

tgacctcctt ggccgcgagc tcggccacgg cctcgcacga aggcgcgaag attcccgctg 1920

gcccttgaag cggaatcacc agcccgacgc ggaactcaac ctcgccgtcc tgcactccag 1980

atcaccgtcg atcccgtgta gtctgcgctt caaagctttc tagcagaaat aattcattct 2040

gaacagaccc cgccgtcgac acgaggagac acccaccatg gccgccggac agcagcgccg 2100

ccccaacctc ctgctgccgt tggtgcgtct gacccacctc gcggagtcgg cgatcgaacg 2160

cgtgctcgcg gactcgtcgc tcaagatcga ggactggcgc gtgctcgacg agttggccgg 2220

acggcgcacc gtgcccatga gcgatctcgc gcaggccacg ctgatcacgg gtccgactct 2280

caccagaacc gtcgatcgcc ttgtgtcgca agggatcatc taccggactg ccgatctgca 2340

tgaccgccgg cgggtgctcg tggcgttgac cccgcggggg cggacgctgc gcaaccgcct 2400

ggtggacgcg gtagccgagg ccgagtgtgc ggcttttgaa tcgtgcgggc tggacgtcga 2460

ccagttgcgc gaactcgtcg acaccacctc gaatttgact tcgtaaccac ccgcgcccgg 2520

cgcgggcgtt cacccttgac ttttattttc atctggatat atttcgggtg aatggaaagg 2580

ggtgaccatg ccgacctaca cattccgttg ttcccactgc ggtcccttcg atctcacctg 2640

cgcgatctcc gagcgcgatg cggcggcgac ctgtccggag tgccggacgc cggcgcgccg 2700

ggtcttcggt tcggtagggc tgacgacatt caccgcggga catcaccgcg cattcgacgc 2760

ggcgtccgcg agcgccgaaa gtcccacggt ggtgaagtcg attcccgcag gcgcggaccg 2820

cccgcgggcc ccgcgccgca atcccggtct accgagtctg ccgaggtact agcgacatgg 2880

gtggcgtcgg gctcttctac gtgggtgcgg tgctcatcat cgacgggctg atgctgctgg 2940

gccgcatcag cccacgaggc gcaacaccgc tgaacttctt cgtcggcgga ctgcaggtgg 3000

tgacgcctac ggtgctgatc ctgcagtccg gcggagacgc ggccgtgatc ttcgcggcct 3060

ccgggctcta cctgttcggc ttcacctacc tgtgggtggc catcaacaac gtgaccgact 3120

gggacggaga aggtctcgga tggttctcgc tgttcgtcgc gatcgccgca ctcggctact 3180

cgtggcacgc gttcaccgcc gaggccgacc cggcgttcgg ggtgatctgg ctgctgtggg 3240

cagtgctgtg gttcatgctg ttcctgctgc tcggcctggg gcacgacgca ctggggcccg 3300

ccgtcgggtt cgtcgcggtg gccgaaggcg tgatcaccgc cgccgtgccg gccttcctga 3360

tcgtgtcggg caactgggaa accggcccgc tccccgccgc ggtcatcgcc gtgatcggtt 3420

ttgccgcagt tgttctcgca taccccatcg ggcgccgtct cgcagcgccg tcagtcacca 3480

accctccacc ggccgcgctc gcggccacca cccgataaga gaaagggagt ccacatatgt 3540

aacggatcca gctgcagaat tcgaagctta tcgatgtcga cgtagttacg agatcggcgg 3600

ccgcatatga gtgtgcccac acaggacgga atgcaccggt tcgtcgacga ggacgtctac 3660

cacgctgacc ggggctcgct gtcggtatcc ggcgcgaagc tgctgttgcc gccgtcgtgt 3720

cccgcgaaat tccgctggga gatggacaac acccggaagc cgaaaaaggt ctgggacttc 3780

ggacatgtcg cgcacaaact ggtgctcggc aagggtgccg agttcgagat cctcgacccc 3840

gaggtgcacg ggctgaaggc ggacggtacg ccgtcggaga agccgaccgc gacgggcatg 3900

tggcgcaagg ccgaggctga ggctcgcaaa cagggcaagg tgccgattca cgtcgacctg 3960

ttcacgaagg cgtacgacat ggccgaaaag gtgcgtcagc acccgacagc cggcccgatc 4020

ttcgccaatc ctgacggcga ggccgaggtc gcgctgtact acaccgaccc cgagaccggc 4080

gtgcggctgc gtggccggat cgactggctc actgacgata tcgatgatta caagacgtcg 4140

atgaccgcga acccggccga gctgaaaacc aagttctaca agctcggcta tttcatgcag 4200

gcggcctggt acatcgatct actggtcgcc ctcgggctcg ccgagaaccc gcgattccgg 4260

ttcatcacgc aggagaaaga accgccctac gtcgtgactc cgatccagta cgacgacgag 4320

gcgatcgaag aggggcggcg ccgcaaccgc caggcgatcc ggctctacgc cgactgcatg 4380

gaatcgggca agtggcctga ctacagcgac gacgtggtca cgatcagcct gccctcgtgg 4440

gggctgccgc gaccgcagac cgtcggcgac gtcgtcaccg acagctatat ctacgacacc 4500

gacccgctcg aagaggccga cccgattgaa ggggattaca tctatggctg aaaatgctgt 4560

caccaagcag gattcgccca aggcacccga gacgatctcg caagtgctgc aggtgctcgt 4620

gccgcagctg gcgcgtgcag tgcccaaggg tatggacccc gaccgcatcg cgcggatcgt 4680

gcaaaccgag atccgcaagt cgcgcaacgc gaaagctgct ggcatcgcta agcagtccct 4740

cgacgactgc acgcaagagt catttgccgg tgcgctgctg acctcggccg cgctcggtct 4800

cgagcccggt gtcaacggcg agtgctacct cgtgccctac cgcgacaccc ggcgcggcgt 4860

ggtcgagtgc cagctgatca tcggctacca gggcatcgtc aaactgttct ggcagcaccc 4920

gcgcgcctcc cggatcgacg cgcagtgggt cggcgcgaac gacgaattcc attacaccat 4980

gggcctcaat ccgacgctga aacacgtgaa ggccaagggt gatcggggta atccggtcta 5040

cttctacgcg atcgtcgagg tgaccggcgc tgagccgctg tgggacgtgt tcaccgccga 5100

cgagatcagg gaattgcgtc gcggcaaggt cggatcctcg ggcgacatca aggacccgca 5160

gcgctggatg gagcggaaga ccgcgctcaa acaggtgctg aagctggcac cgaagacgac 5220

gcggctcgac gcggcgatcc gcgccgacga tcgcccgggc accgacctgt cacagtcgca 5280

ggcgctcgcg ctgccgtcga ccgtcaagcc gacggccgac tacatcgacg gcgagatcgc 5340

cgagccgcac gaggtcgata cgccaccgaa gtcgtcgcgc gcacaacgcg cgcagcgcgc 5400

caccgcgccg gcgcccgacg tgcagatggc caatcccgat cagctgaagc gcctcggcga 5460

gatccagaag gccgaaaagt acaacgacgc cgattggttc aagttcctcg ccgattcggc 5520

cggcgtcaaa gccacgcgcg ccgccgacct cacgttcgac gaggcgaagg ctgtcatcga 5580

catgttcgac gggcccaacg catgagcgcc ccggcgaatt ccgacgcggt ggttgatctg 5640

caactagcgt acgatcgact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 5700

gggcctttcg ttttatgcca tcatggccgc ggtgatcagc tagccacctg acgtcngggg 5760

ggggggaaag ccacgttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata 5820

tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga 5880

gccatattca acgggaaacg tcttgctcga ggccgcgatt aaattccaac atggatgctg 5940

atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc 6000

gcttgtatgg gaagccccat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg 6060

ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc 6120

cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc 6180

ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg 6240

atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta 6300

acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg 6360

atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa 6420

tgcataatct tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg 6480

ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa 6540

tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt 6600

cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc 6660

agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact 6720

ggcagagcat tacgctgact tgacgggacg gcggctttgt tgaataaatc gaacttttgc 6780

tgagttgaag gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca 6840

aaagttcaaa atcaccaact ggtccaccta caacaaagct ctcaccaacc gtggctccct 6900

cactttctgg ctggatgatg gggcgattca ggcctggtat gagtcagcaa caccttcttc 6960

acgaggcaga cctcactagt tccatgagcg tcagaccccg tagaaaagat caaaggatct 7020

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 7080

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 7140

ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 7200

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 7260

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 7320

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 7380

acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgag 7440

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 7500

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 7560

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 7620

aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 7680

gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 7740

cgccgcagcc gaacgaccga gcgcaacgcg tgagcccacc agctccgtaa gttcgggcgc 7800

tgtgtggctc gtacccgcgc attcaggcgg cagggggtct aacgggtcta aggcggcgtg 7860

tacggccgcc acagcggctc tcagcggccc ggaaacgtcc tcgaaacgac gcatgtgttc 7920

ctcctggttg gtacaggtgg ttgggggtgc tcggctgtcg ctggtgttcc accaccaggg 7980

ctcgacggga gagcggggga gtgtgcagtt gtggggtggc ccctcagcga aatatctgac 8040

ttggagctcg tgtcggacca tacaccggtg attaatcgtg gtctactacc aagcgtgagc 8100

cacgtcgccg acgaatttga gcagctctgg ctgccgtact ggccgctggc aagcgacgat 8160

ctgctcgagg ggatctaccg ccaaagccgc gcgtcggccc taggccgccg gtacatcgag 8220

gcgaacccaa cagcgctggc aaacctgctg gtcgtggacg tagaccatcc agacgcagcg 8280

ctccgagcgc tcagcgcccg ggggtcccat ccgctgccca acgcgatcgt gggcaatcgc 8340

gccaacggcc acgcacacgc agtgtgggca ctcaacgccc ctgttccacg caccgaatac 8400

gcgcggcgta agccgctcgc atacatggcg gcgtgcgccg aaggccttcg gcgcgccgtc 8460

gatggcgacc gcagttactc aggcctcatg accaaaaacc ccggccacat cgcctgggaa 8520

acggaatggc tccactcaga tctctacaca ctcagccaca tcgaggccga gctcggcgcg 8580

aacatgccac cgccgcgctg gcgtcagcag accacgtaca aagcggctcc gacgccgcta 8640

gggcggaatt gcgcactgtt cgattccgtc aggttgtggg cctatcgtcc cgccctcatg 8700

cggatctacc tgccgacccg gaacgtggac ggactcggcc gcgcgatcta tgccgagtgc 8760

cacgcgcgaa acgccgaatt tccgtgcaac gacgtgtgtc c 8801

<210> 33

<211> 2145

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 33

ggggaattcg aggacatcag ccggctgccg gatacctgct tcgggatcac cgcggccgac 60

gacgtcgggt atgcgatcgg acagtcactg gttcttgaga tgggcgggga gccgttttgt 120

gtgcgcgaag acgcccgcat cctctaccac gcggcgctgg cccatgcgag caaccacatc 180

gtcaccgtgc tggccgatgc gctcgaggcg ttgcgggccg ccctgagcgg gggggaactg 240

ctcggccaac aaaccgtcga cgaccagccg ggcgggatcg tggagcgcat cgtcgggccg 300

ctggccagag cggcgctgga gaacacgctg cagcggggac aggccgcgct caccggaccg 360

gtcgcccgcg gcgatgcggc agcggtcgcg gatcatctgg cggccctcgc ggacgtcgac 420

gcagcgctgg cccaggcata ccggataaac gcgctgcgga ccgcgcagcg cgcacacgcc 480

cccgcggatg tcgtcgaggt tttgacggcc ttgacataat gtcgcttatc ggcttaatcg 540

atctagaccg gccgtgcgga attaagccgg cccgtaccct gtgaatagag gtccgctgtg 600

acacaagaat ccctgttact tctcgaccgt attgattcgg atgattccta cgcgagcctg 660

cggaacgacc aggagttctg ggagccgctg gcccgccgag ccctggagga gctcgggctg 720

ccggtgccgc cggtgctgcg ggtgcccggc gagagcacca accccgtact ggtcggcgag 780

cccggcccgg tgatcaagct gttcggcgag cactggtgcg gtccggagag cctcgcgtcg 840

gagtcggagg cgtacgcggt cctggcggac gccccggttc cggtgccccg cctcctcggc 900

cgcggcgagc tgcggcccgg caccggagcc tggccgtggc cctacctggt gatgagccgg 960

atgaccggca ccacctggcg gtccgcgatg gacggcacga ccgaccggaa cgcgctgctc 1020

gccctggccc gcgaactcgg ccgggtgctc ggacggctgc acagggtgcc gctgaccggg 1080

aacaccgtgc tcacccccca ttccgaggtc ttcccggaac tgctgcggga acgccgcgcg 1140

gcgaccgtcg aggaccaccg cgggtggggc tacctctcgc cccggctgct ggaccgcctg 1200

gaggactggc tgccggacgt ggacacgctg ctggccggcc gcgaaccccg gttcgtccac 1260

ggcgacctgc acgggaccaa catcttcgtg gacctggccg cgaccgaggt caccgggatc 1320

gtcgacttca ccgacgtcta tgcgggagac tcccgctaca gcctggtgca actgcatctc 1380

aacgccttcc ggggcgaccg cgagatcctg gccgcgctgc tcgacggggc gcagtggaag 1440

cggaccgagg acttcgcccg cgaactgctc gccttcacct tcctgcacga cttcgaggtg 1500

ttcgaggaga ccccgctgga tctctccggc ttcaccgatc cggaggaact ggcgcagttc 1560

ctctgggggc cgccggacac cgcccccggc gcctgatcta gacccgggac ttgacataat 1620

gtcgcttatc ggcttaccgt gctgctggcg attgacgtcc gcaacaccca caccgttgtg 1680

ggcctgctgt ccggaatgaa agagcacgca aaggtcgtgc agcagtggcg gatacgcacc 1740

gaatccgaag tcaccgccga cgaactggca ctgacgatcg acgggctgat cggcgaggat 1800

tccgagcggc tcaccggtac cgccgccttg tccacggtcc cgtccgtgct gcacgaggtg 1860

cggataatgc tcgaccagta ctggccgtcg gtgccgcacg tgctgatcga gcccggagta 1920

cgcaccggga tccctttgct cgtcgacaac ccgaaggaag tgggcgcaga ccgcatcgtg 1980

aactgtttgg ccgcctatga ccggttccgg aaggccgcca tcgtcgttga ctttggatcc 2040

tcgatctgtg ttgatgttgt atcggccaag ggtgaatttc ttggcggcgc catcgcgccc 2100

ggggtgcagg tgtcttccga tgccgcggcg gcccgcaagc ttggg 2145

<210> 34

<211> 1128

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 34

acttgacata atgtcgctta tcggcttaat cgatctagac cggccgtgcg gaattaagcc 60

ggcccgtacc ctgtgaatag aggtccgctg tgacacaaga atccctgtta cttctcgacc 120

gtattgattc ggatgattcc tacgcgagcc tgcggaacga ccaggagttc tgggagccgc 180

tggcccgccg agccctggag gagctcgggc tgccggtgcc gccggtgctg cgggtgcccg 240

gcgagagcac caaccccgta ctggtcggcg agcccggccc ggtgatcaag ctgttcggcg 300

agcactggtg cggtccggag agcctcgcgt cggagtcgga ggcgtacgcg gtcctggcgg 360

acgccccggt tccggtgccc cgcctcctcg gccgcggcga gctgcggccc ggcaccggag 420

cctggccgtg gccctacctg gtgatgagcc ggatgaccgg caccacctgg cggtccgcga 480

tggacggcac gaccgaccgg aacgcgctgc tcgccctggc ccgcgaactc ggccgggtgc 540

tcggacggct gcacagggtg ccgctgaccg ggaacaccgt gctcaccccc cattccgagg 600

tcttcccgga actgctgcgg gaacgccgcg cggcgaccgt cgaggaccac cgcgggtggg 660

gctacctctc gccccggctg ctggaccgcc tggaggactg gctgccggac gtggacacgc 720

tgctggccgg ccgcgaaccc cggttcgtcc acggcgacct gcacgggacc aacatcttcg 780

tggacctggc cgcgaccgag gtcaccggga tcgtcgactt caccgacgtc tatgcgggag 840

actcccgcta cagcctggtg caactgcatc tcaacgcctt ccggggcgac cgcgagatcc 900

tggccgcgct gctcgacggg gcgcagtgga agcggaccga ggacttcgcc cgcgaactgc 960

tcgccttcac cttcctgcac gacttcgagg tgttcgagga gaccccgctg gatctctccg 1020

gcttcaccga tccggaggaa ctggcgcagt tcctctgggg gccgccggac accgcccccg 1080

gcgcctgatc tagacccggg acttgacata atgtcgctta tcggctta 1128

<210> 35

<211> 3879

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 35

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420

tgcatctaga tatcggatcc cgggatgcat cagatctctc gagacttgac ataatgtcgc 480

ttatcggctt aatcgatcta gaccggccgt gcggaattaa gccggcccgt accctgtgaa 540

tagaggtccg ctgtgacaca agaatccctg ttacttctcg accgtattga ttcggatgat 600

tcctacgcga gcctgcggaa cgaccaggag ttctgggagc cgctggcccg ccgagccctg 660

gaggagctcg ggctgccggt gccgccggtg ctgcgggtgc ccggcgagag caccaacccc 720

gtactggtcg gcgagcccgg cccggtgatc aagctgttcg gcgagcactg gtgcggtccg 780

gagagcctcg cgtcggagtc ggaggcgtac gcggtcctgg cggacgcccc ggttccggtg 840

ccccgcctcc tcggccgcgg cgagctgcgg cccggcaccg gagcctggcc gtggccctac 900

ctggtgatga gccggatgac cggcaccacc tggcggtccg cgatggacgg cacgaccgac 960

cggaacgcgc tgctcgccct ggcccgcgaa ctcggccggg tgctcggacg gctgcacagg 1020

gtgccgctga ccgggaacac cgtgctcacc ccccattccg aggtcttccc ggaactgctg 1080

cgggaacgcc gcgcggcgac cgtcgaggac caccgcgggt ggggctacct ctcgccccgg 1140

ctgctggacc gcctggagga ctggctgccg gacgtggaca cgctgctggc cggccgcgaa 1200

ccccggttcg tccacggcga cctgcacggg accaacatct tcgtggacct ggccgcgacc 1260

gaggtcaccg ggatcgtcga cttcaccgac gtctatgcgg gagactcccg ctacagcctg 1320

gtgcaactgc atctcaacgc cttccggggc gaccgcgaga tcctggccgc gctgctcgac 1380

ggggcgcagt ggaagcggac cgaggacttc gcccgcgaac tgctcgcctt caccttcctg 1440

cacgacttcg aggtgttcga ggagaccccg ctggatctct ccggcttcac cgatccggag 1500

gaactggcgc agttcctctg ggggccgccg gacaccgccc ccggcgcctg atctagaccc 1560

gggacttgac ataatgtcgc ttatcggctt actcgagatt atccatggcg gccgcactag 1620

tctgcagagg cctgcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 1680

attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 1740

ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 1800

agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 1860

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 1920

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 1980

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 2040

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 2100

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 2160

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 2220

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 2280

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 2340

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 2400

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 2460

agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 2520

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 2580

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 2640

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 2700

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 2760

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 2820

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 2880

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 2940

gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 3000

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 3060

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 3120

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 3180

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 3240

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 3300

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 3360

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 3420

cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 3480

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 3540

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 3600

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 3660

ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 3720

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 3780

cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 3840

taacctataa aaataggcgt atcacgaggc cctttcgtc 3879

<210> 36

<211> 4891

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic polynucleotides

<400> 36

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420

tgcatctaga tatcggatcc cgggatgcat cagatctctc gaggaattcg aggacatcag 480

ccggctgccg gatacctgct tcgggatcac cgcggccgac gacgtcgggt atgcgatcgg 540

acagtcactg gttcttgaga tgggcgggga gccgttttgt gtgcgcgaag acgcccgcat 600

cctctaccac gcggcgctgg cccatgcgag caaccacatc gtcaccgtgc tggccgatgc 660

gctcgaggcg ttgcgggccg ccctgagcgg gggggaactg ctcggccaac aaaccgtcga 720

cgaccagccg ggcgggatcg tggagcgcat cgtcgggccg ctggccagag cggcgctgga 780

gaacacgctg cagcggggac aggccgcgct caccggaccg gtcgcccgcg gcgatgcggc 840

agcggtcgcg gatcatctgg cggccctcgc ggacgtcgac gcagcgctgg cccaggcata 900

ccggataaac gcgctgcgga ccgcgcagcg cgcacacgcc cccgcggatg tcgtcgaggt 960

tttgacggca cttgacataa tgtcgcttat cggcttaatc gatctagacc ggccgtgcgg 1020

aattaagccg gcccgtaccc tgtgaataga ggtccgctgt gacacaagaa tccctgttac 1080

ttctcgaccg tattgattcg gatgattcct acgcgagcct gcggaacgac caggagttct 1140

gggagccgct ggcccgccga gccctggagg agctcgggct gccggtgccg ccggtgctgc 1200

gggtgcccgg cgagagcacc aaccccgtac tggtcggcga gcccggcccg gtgatcaagc 1260

tgttcggcga gcactggtgc ggtccggaga gcctcgcgtc ggagtcggag gcgtacgcgg 1320

tcctggcgga cgccccggtt ccggtgcccc gcctcctcgg ccgcggcgag ctgcggcccg 1380

gcaccggagc ctggccgtgg ccctacctgg tgatgagccg gatgaccggc accacctggc 1440

ggtccgcgat ggacggcacg accgaccgga acgcgctgct cgccctggcc cgcgaactcg 1500

gccgggtgct cggacggctg cacagggtgc cgctgaccgg gaacaccgtg ctcacccccc 1560

attccgaggt cttcccggaa ctgctgcggg aacgccgcgc ggcgaccgtc gaggaccacc 1620

gcgggtgggg ctacctctcg ccccggctgc tggaccgcct ggaggactgg ctgccggacg 1680

tggacacgct gctggccggc cgcgaacccc ggttcgtcca cggcgacctg cacgggacca 1740

acatcttcgt ggacctggcc gcgaccgagg tcaccgggat cgtcgacttc accgacgtct 1800

atgcgggaga ctcccgctac agcctggtgc aactgcatct caacgccttc cggggcgacc 1860

gcgagatcct ggccgcgctg ctcgacgggg cgcagtggaa gcggaccgag gacttcgccc 1920

gcgaactgct cgccttcacc ttcctgcacg acttcgaggt gttcgaggag accccgctgg 1980

atctctccgg cttcaccgat ccggaggaac tggcgcagtt cctctggggg ccgccggaca 2040

ccgcccccgg cgcctgatct agacccggga cttgacataa tgtcgcttat cggcttaccg 2100

tgctgctggc gattgacgtc cgcaacaccc acaccgttgt gggcctgctg tccggaatga 2160

aagagcacgc aaaggtcgtg cagcagtggc ggatacgcac cgaatccgaa gtcaccgccg 2220

acgaactggc actgacgatc gacgggctga tcggcgagga ttccgagcgg ctcaccggta 2280

ccgccgcctt gtccacggtc ccgtccgtgc tgcacgaggt gcggataatg ctcgaccagt 2340

actggccgtc ggtgccgcac gtgctgatcg agcccggagt acgcaccggg atccctttgc 2400

tcgtcgacaa cccgaaggaa gtgggcgcag accgcatcgt gaactgtttg gccgcctatg 2460

accggttccg gaaggccgcc atcgtcgttg actttggatc ctcgatctgt gttgatgttg 2520

tatcggccaa gggtgaattt cttggcggcg ccatcgcgcc cggggtgcag gtgtcttccg 2580

atgccgcggc ggcccgcaag cttctcgaga ttatccatgg cggccgcact agtctgcaga 2640

ggcctgcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 2700

ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 2760

taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 2820

aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 2880

attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2940

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 3000

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 3060

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 3120

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 3180

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 3240

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 3300

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 3360

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 3420

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 3480

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 3540

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3600

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3660

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3720

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3780

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 3840

ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 3900

ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 3960

atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 4020

ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 4080

tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 4140

attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 4200

tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 4260

ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 4320

gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 4380

gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 4440

gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 4500

aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 4560

taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 4620

tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 4680

tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 4740

atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 4800

tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 4860

aaaaataggc gtatcacgag gccctttcgt c 4891

<210> 37

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 37

ggggactagt aacacccctt gtattactg 29

<210> 38

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 38

ggggtgatca tcagaattgg ttaattggtt g 31

<210> 39

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 39

ggggactagt acgaccagcc g 21

<210> 40

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 40

ggggtgatca gctatcccac accg 24

<210> 41

<211> 935

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<400> 41

atgacgattc ctgcgttcca tcccggtgaa ctcaatgtgt actcggcacc gggggatgtc 60

gccgatgtca gtcgcgcact gcgactcacc ggccggcgag tgatgttggt gcctactatg 120

ggtgcgctgc acgaaggcca cctcgcgttg gtgcgtgcgg ccaagcgggt gcccggatcg 180

gtcgtcgtcg tgtcgatctt cgtcaacccg atgcaattcg gtgccgggga agatctcgac 240

gcctatcccc gcaccccgga cgacgacctg gcgcaactgc gggccgaagg cgtggaaatc 300

gctttcacgc caactaccgc ggcgatgtat cccgacggcc tgcgcaccac cgtgcaaccc 360

ggtccgttgg ccgccgaact cgagggcggc ccgcggccaa cccatttcgc cggcgtgctg 420

acggtcgtgc taaagctgct gcagatcgtg cgcccggatc gggtgttctt cggtgagaag 480

gactaccagc agctggtgct gatccggcag ctggtcgcgg acttcaacct cgatgtcgcg 540

gtggtcggcg tgccgaccgt gcgcgaagcc gacgggctgg cgatgtcgtc gcgcaaccgc 600

tacctggacc cggcccagcg tgcggcggcc gtcgcgctct cggcggcgct aacggccgca 660

gcgcatgcgg caacggctgg cgcgcaggcc gcgctggatg ccgcccgtgc ggtgctcgac 720

gctgcacccg gcgtggcgtg gcggtcgact acctggagct gcgcgatatc gggcttggcc 780

cgatgccgct caacggttcc ggtcggctgc tggttgctgc ccggcttggc accaccaggc 840

tgctggacaa cattgcgatt gaaatcggaa ctttcgccgg caccgaccgc ccggacggat 900

accgggcaat cctcgaatca cattggagaa actga 935

<210> 42

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 42

gggacgcgtt attgatcgct gatggtcgat t 31

<210> 43

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 43

gagaaaactc accgaggcag 20

<210> 44

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 44

gtatttcgtc tcgctcaggc 20

<210> 45

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 45

gcgatggtgg cttctccctc g 21

<210> 46

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 46

ccatcttgca cagctcgcgt ag 22

<210> 47

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 47

taaggccaac cgtgaaaaga tg 22

<210> 48

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 48

ctggatggct acgtacatgg ct 22

<210> 49

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 49

gaccctcaca ctcagatcat c 21

<210> 50

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 50

gctgctcctc cacttggt 18

<210> 51

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 51

ccacagccct ctccatcaac 20

<210> 52

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 52

ctccgtcatc tccataggga 20

<210> 53

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 53

ctgcaagaga cttccatcca g 21

<210> 54

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 54

caggtctgtt gggagtgg 18

<210> 55

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 55

agcggctgac tgaactcaga ttgt 24

<210> 56

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 56

gtcacagttt tcagctgtat aggg 24

<210> 57

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 57

ggagagtgtg gatcccaa 18

<210> 58

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 58

gtggagtttg agtctgcag 19

<210> 59

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 59

ggctcagcca gatgcagtta ac 22

<210> 60

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 60

gatcctcttg tagctctcca gc 22

<210> 61

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 61

gaaagacgtt tatgttgtag agg 23

<210> 62

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 62

gactccatgt ctctggtctg 20

<210> 63

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 63

ggagttcgag gaaccctagt g 21

<210> 64

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 64

gggatttgta gtggatcgtg c 21

<210> 65

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 65

gtgggactca agggatccct ctc 23

<210> 66

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 66

gcttccctat ggccctcatt c 21

<210> 67

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 67

gttctcagcc caacaataca ag 22

<210> 68

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 68

ggaacattct gtgctgtccc 20

<210> 69

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 69

ctctgatgca ggtccctatg gtg 23

<210> 70

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 70

ggcagagggt gacggatgta g 21

<210> 71

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 71

ctcgcttcgg cagcacatat ac 22

<210> 72

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 72

aatatggaac gcttcacgaa tttg 24

<210> 73

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 73

caacttgctt ggattcctac aaag 24

<210> 74

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 74

tattcaagcc tcccattcaa ttg 23

<210> 75

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 75

ggtacatcct cgacggcatc t 21

<210> 76

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 76

gtgcctcttt gctgctttca c 21

<210> 77

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 77

caggattcat gtgccagggt 20

<210> 78

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 78

ccaaagacca catgcttgcc 20

<210> 79

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 79

gagtcttcac actcctggc 19

<210> 80

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 80

gtccttcagg catgagacag 20

<210> 81

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 81

gcgttcctgc tgtgcttctc 20

<210> 82

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 82

ccattcagct gcctcaggag c 21

<210> 83

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 83

cgtcttggtt ttgcagctct 20

<210> 84

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 84

cgtccttttg ccagttcctc 20

<210> 85

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 85

ggtgagggga ctggactttt ag 22

<210> 86

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 86

ttgttgggct gggaatagca 20

<210> 87

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 87

tccacctccc tttacccagt 20

<210> 88

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 88

agagctagga gagccgtcat 20

<210> 89

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 89

caggtctctg tcacgcttct g 21

<210> 90

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 90

gccagtgaat gagtagcagc ag 22

<210> 91

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 91

acaagcgcac cctctgttac 20

<210> 92

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 92

ggtcaggaaa atgacacccg 20

<210> 93

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 93

gacttcacca tggaacccgt 20

<210> 94

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 94

ggagactgcc cattctcgac 20

<210> 95

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 95

cgtccctctc atacactgg 19

<210> 96

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 96

catgctttcc gtgctcatg 19

<210> 97

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 97

tgacgtcact ggagttgtcc 20

<210> 98

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 98

cctcgacgtt tgggactgat 20

<210> 99

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 99

cctctggata cagctgcgac 20

<210> 100

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 100

tgccgggtgg ttcaattttt c 21

<210> 101

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 101

tactagcggt tttacgggcg 20

<210> 102

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 102

tcgaacagga ggagcagaga gcga 24

<210> 103

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 103

cggtgaagaa tggatgacct 20

<210> 104

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 104

aaacgagacc cttgcacaac 20

<210> 105

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 105

atcagtcagt ggcccagaag accc 24

<210> 106

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 106

ccacgtcccg gatcatgctt cag 23

<210> 107

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthetic primers

<400> 107

gggcatcata tgcacgctgt gactcgtc 28

Claims (16)

1. A method of inhibiting the expression of Myeloid Derived Suppressor Cells (MDSCs), M2 macrophages and Treg cells and inducing the expression of macrophages, dendritic Cells (DCs) and T effector cells in a tumor, the method comprising the steps of:

administering to a subject having a tumor a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a protein that produces a STING agonist, or a functional portion thereof;

inhibiting the expression of MDSC, M2 macrophages and Treg cells in the tumor; and

inducing expression of macrophages, dendritic Cells (DCs) and T effector cells in the tumor, thereby inhibiting expression of MDSC, M2 macrophages and Treg cells, and inducing expression of macrophages, DCs and T effector cells in the tumor.

2. The method of claim 1, wherein inhibition of expression of MDSCs, M2 macrophages, and Treg cells in a tumor is observed when compared to expression of MDSCs, M2 macrophages, and Treg cells in a tumor of a reference subject that has not been administered a pharmaceutical composition comprising the mycobacterium strain.

3. The method of claim 1, wherein inducing expression of macrophages, dendritic Cells (DCs) and T effector cells in a tumor is observed when compared to expression of macrophages, dendritic Cells (DCs) and T effector cells in a tumor of a reference subject that has not been administered a pharmaceutical composition comprising said mycobacterium strain.

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 combinations 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 an Rv1354c protein or functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (DisA) protein or a functional portion thereof, and combinations thereof.

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

7. The method according to claim 6, wherein the tumor is non-muscle invasive bladder cancer and BCG non-responsive non-muscle invasive bladder cancer (BCG non-responsive NMIBC) and the pharmaceutical composition is administered by intravesical instillation.

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

9. The method of claim 6, wherein the tumor is an epithelial cancer selected from the group consisting of: colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngeal cancer, bronchial cancer, and combinations thereof, and the pharmaceutical composition is administered to the 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 injection, intravenous injection, intradermal injection, transdermal injection, intravesical injection, topical injection, intramuscular injection, 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), cimiraprizumab (anti-PD-1 antibody), astuzumab (anti-PD-L1 antibody), avizumab (anti-PD-L1 antibody), de-wauzumab (anti-PD-L1 antibody), and combinations thereof.

13. The method of claim 1, wherein the induced macrophage is an M1 macrophage.

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 injection, intradermal injection, transdermal injection, intravesical injection, topical injection, intramuscular injection, or subcutaneous injection.

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