Double and triple knock-out vaccine compositions against tuberculosis

Abstract

Provided here are nucleic acid constructs containing a Mycobacterium tuberculosis genome comprising a mutation (such as a deletion) of the sigH gene (AsigH) and a mutation (such as a deletion) of at least one additional gene, and mutant Mycobacterium tuberculosis encoded by the nucleic acid constructs. Also provided are methods of making such nucleic acid constructs and Mycobacterium tuberculosis mutants, and uses thereof. The construct can stimulate an immune response against Mycobacterium tuberculosis in a subject, and can be used as live attenuated vaccines.

Description

DOUBLE AND TRIPLE KNOCK-OUT VACCINE COMPOSITIONS AGAINST TUBERCULOSISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 701,515 filed on September 30, 2024, the content of which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under R01AI138587 and R01AI134240 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates to live-attenuated Mycobacterium tuberculosis (Mtb) vaccines, compositions, methods for making such compositions, and methods of use of such compositions.BACKGROUND

[0004] Tuberculosis (TB) is an infectious disease generally caused by Mycobacterium tuberculosis (Mtb). Tuberculosis predominantly affects the lungs. Around 10% of latent TB progress to active disease that, if left untreated, kill about half of those affected. Typical symptoms of active TB are chronic cough with blood-containing mucus, fever, night sweats, and weight loss. Active TB is a highly infectious airborne disease.

[0005] Currently, Bacillus Calmette-Guérin (BCG) is the only licensed vaccine to protect against TB, yet its efficacy has been called into question as approximately 10 million new cases of TB occur in BCG-vaccinated people, leading to an average of 1.5M deaths annually including those in Group of 20 (G20) countries such India, Russia, China, South Africa and Indonesia. Of the vaccines under development, most use subunit proteins and the durability of immune responses generated by them is questionable.SUMMARY

[0006] The development of new and efficacious TB vaccines is urgently needed to combat tuberculosis, co-infection of tuberculosis and another microbe, or associated conditions. Attenuated, live-replicating Mtb are most likely to afford long-lasting protection through long-lived immune responses, since these Mtbs express the full complement of protective antigens not present in BCG and other classes of vaccines.1

[0007] Provided here are nucleic acid constructs containing a Mycobacterium tuberculosis genome comprising a mutation (such as a deletion) of the sigH gene (ΔsigH) and a mutation (such as a deletion) of at least one additional gene, and mutant Mycobacterium tuberculosis encoded by the nucleic acid constructs, such as ΔsigH double and triple knockout (DKO / TKO) Mtb mutant strains that represent rationally attenuated and live-replicating Mtb vaccine candidates, which adhere to Geneva consensus safety requirements. Also provided are methods of making such nucleic acid constructs and Mycobacterium tuberculosis mutants, and uses thereof. The construct can stimulate an immune response against Mycobacterium tuberculosis in a subject, and can be used as live attenuated vaccines.

[0008] In one aspect, provided is a nucleic acid construct containing a Mycobacterium tuberculosis genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene.

[0009] In embodiments, Mycobacterium tuberculosis is Mycobacterium tuberculosis CDC1551. The nucleic acid construct contains a Mycobacterium tuberculosis CDC1551 genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene.

[0010] In some embodiments, the mutation in the sigH gene is a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene is a deletion of the at least one additional gene.

[0011] In some embodiments, the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

[0012] In some embodiments, the deletion of at least one additional gene contains: a deletion of fbpA (ΔfbpA); a deletion of sapM (ΔsapM); a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM); a deletion of mce4E (Δmce4E); a deletion of mce4F (Δmce4F); a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F), a deletion of secA2 (ΔsecA2); a deletion of sodA (ΔsodA); a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA); a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC)); a deletion of Rv3683 (ΔRv3683); a deletion of leuD (ΔleuD); a deletion of panCD (ΔpanCD); a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD); a deletion of metA (ΔmetA); a deletion of cobM (ΔcobM); a deletion of mce1A (Δmce1A); a deletion of fadD29 (ΔfadD29); or a combination of any thereof.2

[0013] In one aspect, provided is a mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome containing a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene. In some embodiments, the mutant Mycobacterium tuberculosis is non-pathogenic.

[0014] In some embodiments, the mutation in the sigH gene is a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene is a deletion of the at least one additional gene.

[0015] In some embodiments, the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

[0016] In some embodiments, the deletion of at least one additional gene contains: a deletion of fbpA (ΔfbpA); a deletion of sapM (ΔsapM); a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM); a deletion of mce4E (Δmce4E); a deletion of mce4F (Δmce4F); a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F), a deletion of secA2 (ΔsecA2); a deletion of sodA (ΔsodA); a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA); a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC)); a deletion of Rv3683 (ΔRv3683); a deletion of leuD (ΔleuD); a deletion of panCD (ΔpanCD); a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD); a deletion of metA (ΔmetA); a deletion of cobM (ΔcobM); a deletion of mce1A (Δmce1A); a deletion of fadD29 (ΔfadD29); or a combination of any thereof..

[0017] In one aspect, provided is an immunogenic composition containing the mutant Mycobacterium tuberculosis provided herein.

[0018] In one aspect, provided is a method of stimulating an immune response against Mycobacterium tuberculosis in a subject. The method includes administering an effective amount of the mutant Mycobacterium tuberculosis or the immunogenic composition provided herein. In some embodiments, the method includes stimulating an immune response against Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infection.

[0019] In one aspect, provided is a method of treating tuberculosis in a subject. The method includes administering an effective amount of an immunogenic composition containing a mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome containing a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene. In some embodiments, the mutant Mycobacterium tuberculosis is non-pathogenic.3

[0020] In some embodiments, the mutation in the sigH gene is a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene is a deletion of the at least one additional gene.

[0021] In some embodiments, the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

[0022] In some embodiments, the deletion of at least one additional gene contains: a deletion of fbpA (ΔfbpA); a deletion of sapM (ΔsapM); a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM); a deletion of mce4E (Δmce4E); a deletion of mce4F (Δmce4F); a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F), a deletion of secA2 (ΔsecA2); a deletion of sodA (ΔsodA); a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA); a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC)); a deletion of Rv3683 (ΔRv3683); a deletion of leuD (ΔleuD); a deletion of panCD (ΔpanCD); a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD); a deletion of metA (ΔmetA); a deletion of cobM (ΔcobM); a deletion of mce1A (Δmce1A); a deletion of fadD29 (ΔfadD29); or a combination of any thereof.

[0023] In some embodiments, the subject has or is at risk of developing tuberculosis and human immunodeficiency virus (TB / HIV) co-infection, and the method provided herein can treat or prevent TB / HIV co-infection.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. To facilitate this description, like reference numerals designate like structural elements or procedures in a method. Embodiments are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings.

[0026] FIG.1 depicts a schematic representation for the generation of allelic exchange sequence (AES) for gene deletions in Mtb.

[0027] FIG.2A depicts the colony forming units (CFUs) in lung lysates from TB infected rhesus macaques that were either unvaccinated (red) or vaccinated with BCG (light blue) or MtbΔsigH (dark blue) represented as CFU per gram of lung tissue. FIG 2B depicts the colony forming units (CFUs)4from lysates from the bronchial lymph nodes (LN) from TB infected rhesus macaques that were either unvaccinated (red) or vaccinated with BGC (light blue) or MtbΔsigH (dark blue) represented as CFU per gram of bronchial LN tissue. * denotes p<0.05, ** p<0.01, **** p<0.0001. Statistical significance was determined via ANOVA. FIG 2C represents immunofluorescent staining (left) of CD20 (red), CD3 (green) and DAPI (blue) and hematoxylin and eosin staining (right) of lung sections from TB infected rhesus macaques that were either untreated (top), BCG vaccinated (middle), or MtbΔsigH vaccinated (bottom).

[0028] FIG. 3A depicts the Log10transformed CFU per gram of lung tissue in TB infected unvaccinated cynomolgus macaques and TB infected cynomolgus macaques vaccinated with either BCG or MtbΔsigH via aerosol inhalation. P-values are displayed above the bar graphs. P-values were determined via ANOVA. FIG.3B displays the number of lung lobes that were either sterile (blue) or non-sterile (magenta) in TB infected cynomolgus macaques that were either unvaccinated, vaccinated with BCG, or vaccinated with MtbΔsigH (bar graph). ** denotes p<0.01, **** p<0.0001. Pie chart (right) depicting the % of sterile (blue) and non-sterile (magenta) lung lobes in TB infected cynomolgus macaques that were either unvaccinated or vaccinated with BCG or MtbΔsigH. FIG 3C represents gross pathological features of the lungs from TB infected cynomolgus macaques that were either unvaccinated or received aerosol vaccination of BCG or MtbΔsigH. Arrows indicate tuberculosis lesions.

[0029] FIG.4A is a list of proposed vaccine candidates along with their corresponding Rv number and CDC 1551 entry.

[0030] FIG.4B1 schematically depicts gene locus and FIG.4B2 depicts PCR-based analysis in the wild-type and metA deletion strain of M. tuberculosis CDC1551.

[0031] FIG.4C1 schematically depicts gene locus and FIG.4C2 depicts PCR-based analysis in the wild-type and leuD deletion strain of M. tuberculosis CDC1551.

[0032] FIG.4D1 schematically depicts gene locus and FIG.4D2 depicts PCR-based analysis in the wild-type and hadC deletion strain of M. tuberculosis CDC1551.

[0033] FIG.4E1 schematically depicts gene locus and FIG.4E2 depicts PCR-based analysis in the wild-type and MT3785 deletion strain of M. tuberculosis CDC1551.

[0034] FIG.4F1 schematically depicts gene locus and FIG.4F2 depicts PCR-based analysis in the wild-type and mce4E-mce4F deletion strain of M. tuberculosis CDC1551.5

[0035] FIG.4G1 schematically depicts gene locus and FIG.4G2 depicts PCR-based analysis in the wild-type and mce1A deletion strain of M. tuberculosis CDC1551.

[0036] FIG.4H1 schematically depicts gene locus and FIG.4H2 depicts PCR-based analysis in the wild-type and secA2 deletion strain of M. tuberculosis CDC1551.

[0037] FIG.4I1 schematically depicts gene locus and FIGs.4I2 and 4I3 depicts PCR-based analysis in the wild-type and sapM and fbpA deletion strain of M. tuberculosis CDC1551. FIG. 4J1 schematically depicts gene locus and FIG.4J2 depicts PCR-based analysis in the wild-type and sigH deletion strain of M. tuberculosis CDC1551. The open reading frames of metA (FIG.4B1), leuD (FIG. 4C1), hadC (FIG.4D1), MT3785 (FIG.4E1), mce4E-mce4F (FIG.4F1), mce1A (FIG.4G1), secA2 (FIG.4H1) and sapM (FIG.4I1), were separately replaced with the hygromycin resistance gene (hygR) in the M. tuberculosis genome. MtbΔmce1A and MtbΔsecA2 were also unmarked in another phage transduction step using the temperature-sensitive mycobacteriophage phAE280. In the double mutant strain, MtbΔsapMΔfbpA, the open reading frames of fbpA was replaced with kanamycin resistance gene (kanR) in the genome of the MtbΔsapM strain (FIG.4I1). sigH was replaced with apramycin resistance gene (apraR) in each of the eight mutant strains. The disruptions of various genes, in their respective single and double mutant strain were confirmed by PCR amplification using locus-specific primers. The solid black arrows depict the region of binding by the primers for PCR-based screening. The lanes presented in each panel are derived from the same gel, with images cropped for clarity.

[0038] FIG.5A is a schematic representation of cloning strategy to generate single gene knockouts in Mtb. FIG.5B represents images from gel electrophoresis of polymerase chain reaction (PCR) reactions using the HR-F and HR-R primers depicted in Fig. 5A. Wild-type Mtb strains show a product of ~1.5kb, whereas knockout (KO) Mtb strains show a PCR product ~4kb in size. FIG.5C depict gel electrophoresis of PCR products using hygF and HR-R primers depicted in Fig.5A. KO Mtb strains show a PCR product ~1.5kb in size corresponding to the hygromycin resistant insert which is absent in the WT Mtb strain. FIG.5D depicts gel electrophoresis images of PCR products amplified from KO Mtb strains that underwent an additional transduction to remove the hygromycin and SacB selection markers. Primers directed towards the sacB region (left) or hygromycin resistance region (right) show no PCR products for the unmarked mutants (orange arrows) whereas the marked KO mutants show products around 1.2kb and 250bp respectively. FIG.5E depicts PCR confirmation for sigH gene deletion in mce1 knockout-unmarked strain. The PCR product corresponds to the size6of WT sigH and sigH KO (2.2 kb in KO; 855 bp in WT), indicating integration of the ^^^^ sequences into the genome of the sigH KO strain.

[0039] FIG.6A depicts longitudinal measurements of bacterial burden in bronchoalveolar lavage (BAL) fluid from rhesus macaques challenged with Mtb knockout (KO) strains via aerosol and subsequently co-infected with SIV intravenously. The dashed line indicates the time of SIV challenge.

[0040] FIG.6B depicts endpoint BAL colony-forming units (CFUs) at necropsy, comparing Mtb KOs / SIV (lavender) co-infected animals with previously studied Mtb / SIV (blue) and MtbΔsigH / SIV (turquoise) cohorts.

[0041] FIG.6C depicts total bacterial burden in lung tissue at necropsy across the same groups.

[0042] FIG.6D depicts bacillary loads specifically within lung granulomas.

[0043] FIG.6E depicts bacterial burden in bronchial lymph nodes (BrLN).

[0044] FIGs.6F–6M depict strain-specific bacterial burden determined by PCR analysis of lysates from colonies isolated from BAL fluid, lung tissue, and BrLN collected at necropsy. Data for Mtb / SIV and MtbΔsigH / SIV co-infected macaques were obtained from previously published studies, as these groups were not included in the current experimental cohort. In FIGs.6A-6M, Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparisons test in GraphPad Prism version 9.2.0 for macOS. A P value of <0.05 was considered statistically significant. Significance levels are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are presented as Mean ± SEM.

[0045] FIG. 7A depicts computed tomography (CT) imaging of the thoracic region in rhesus macaques at 4 weeks post-challenge with Mtb knockout (KO) strains, illustrating early lung pathology.

[0046] FIG. 7B depicts CT imaging of the same animals at 4 weeks post-SIV co-infection, highlighting changes in TB lesion development prior to necropsy.

[0047] FIG.7C depicts representative gross pathology of lung tissue from macaques euthanized at 4 weeks post-Mtb KO challenge, showing minimal visible lesions compared to historical controls.

[0048] FIG.7D depicts representative gross pathology of lung tissue from macaques euthanized at 4 weeks post-SIV co-infection, demonstrating preserved lung architecture.7

[0049] FIG.7E depicts a representative high-resolution photomicrograph of H&E-stained lung tissue collected at necropsy, with granulomatous lesions (2–4 mm) marked by arrows.

[0050] FIG. 7F depicts H&E-stained lung sections from Mtb KOs / SIV co-infected macaques showing granuloma in Mtb / SIV co-infected control animal and iBALT / iBALT-like structure in Mtb KOs / SIV co-challenged animal.

[0051] FIG. 7G depicts quantification of percentage lung involvement, calculated by a board-certified pathologist based on lesion counts per lung lobe.

[0052] FIG.7H depicts representative immunohistochemistry-stained lung sections from Mtb KO-infected macaques, highlighting iBALT markers: CD20 (purple), CD3 (teal), and CD68 (green).

[0053] FIG.7I depicts similar immunohistochemistry staining in lung sections from Mtb KOs / SIV co-infected macaques, showing organized lymphoid structures. In FIGs.7A-7I, Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparisons test in GraphPad Prism v9.2.0. A P value of <0.05 was considered statistically significant. Significance levels are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are presented as Mean ± SEM.

[0054] FIG.8A depicts the frequency of antigen-specific multifunctional CD4⁺ T cells expressing IFN-γ and TNF-α in BAL fluid collected 4 weeks post-Mtb KO challenge.

[0055] FIG.8B depicts the frequency of CD4⁺ T cells co-expressing IFN-γ, TNF-α, and IL-2 under the same conditions.

[0056] FIG.8C depicts the frequency of CD8⁺ T cells expressing IFN-γ and TNF-α in BAL fluid collected 4 weeks post-Mtb KO challenge.

[0057] FIG.8D depicts the frequency of CD8⁺ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the same samples.

[0058] FIG.8E depicts the frequency of multifunctional CD8⁺ T cells expressing IFN-γ and TNF-α in BAL fluid collected 8 weeks post-Mtb KO challenge (i.e., 4 weeks post-SIV co-infection).

[0059] FIG.8F depicts the frequency of CD8⁺ T cells co-expressing IFN-γ, TNF-α, and IL-2 at the same time point.

[0060] FIG.8G depicts the frequency of antigen-specific CD4⁺ T cells expressing IFN-γ in BAL fluid collected 3–4 weeks post-challenge.8

[0061] FIG.8H depicts CD4⁺ T cells expressing TNF-α, and FIG.8I depicts those expressing IL-17.

[0062] FIG.8J depicts CD8⁺ T cells expressing IFN-γ, FIG.8K depicts those expressing TNF-α, and FIG.8L depicts those expressing IL-17.

[0063] Data shown in FIGs.8G–8L compare responses among Mtb KO-infected (teal), Mtb WT-infected (black), and naïve (orange) macaques. BAL cells were stimulated overnight (16–18 h) with either whole-cell or cell wall fractions of Mtb, or left unstimulated. Activated cells were stained with fluorophore-tagged antibodies, and surface marker expression was analyzed via flow cytometry. In FIGs. 8A-8L, Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparisons test in GraphPad Prism v9.2.0. A P value of < 0.05 was considered statistically significant. Significance levels are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are presented as Mean ± SEM (n = 3).DETAILED DESCRIPTIONA. Definitions

[0064] The present disclosure describes various embodiments related to compositions and methods for prevention or treatment of tuberculosis. In the following description, numerous details are set forth in order to provide a thorough understanding of the various embodiments. Before the present methods and compositions are described, it is to be understood that these embodiments are not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present embodiments will be limited only by the appended claims. The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous.

[0065] An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect desired results (such as desired clinical results, such as disease prevention, to achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations.

[0066] “Administering” refers to the physical introduction of a therapeutic agent to a subject in need thereof. Example routes of administration for agents to inhibit proliferation of mesenchymal cancer stem cells. include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other9parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. A therapeutic agent may be administered via a non-parenteral route, or orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. Therapeutic agents can be constituted in a composition, such as an immunogenic composition containing an antibody and a pharmaceutically acceptable carrier.

[0067] As used herein, an “immunogenic composition” is a substance that can cause an immune response in the body. Immunogenicity is the ability of a substance to trigger an immune response, which can be either humoral or cellular. An immunogenic composition can include an active component and a pharmaceutically acceptable carrier.

[0068] As used herein, “carrier” or “excipient” refers to an inert compound that is compatible with any other ingredients in the formulation and is not deleterious to the active compound (for example, mutant Mycobacterium tuberculosis) or a subject that the formulation is administered thereto. Suitable carriers can be added to improve recovery, efficacy, or physical properties and / or to aid in packaging and administration. Such carriers may be added individually or in combination. Non-limiting examples of carriers include inert diluents (for example, sodium and calcium carbonate, sodium and calcium phosphate, and lactose), disintegrating agents (for example, corn starch, alginic acid), binding agents (for example, starch, gelatin), lubricating agents (for example, magnesium stearate, stearic acid, talc), sweetening agents, flavoring agents, coloring agents, preservatives, coating agents (for example, glyceryl monostearate, glyceryl distearate).

[0069] As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0070] A “subject” refers an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey) and a non-primate (such as a mouse). In some aspects of the10disclosure, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.

[0071] A “patient” refers to a subject who shows symptoms and / or signs of a disease, is under treatment for disease, has been diagnosed with a disease, and / or is at risk of developing a disease. A “patient” can be a human or veterinary subject. Any reference to subjects in the present disclosure should be understood to include the possibility that the subject is a “patient” unless clearly dictated otherwise by context. More specifically, the subject in certain aspects is a patient who has or at risk of having tuberculosis.

[0072] As used herein, the terms “treatment,” “treating,” and “treat” refer to any indicia of success in the treatment or amelioration of an injury, disease, or condition (such as tuberculosis), including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the injury, disease, or condition more tolerable to the subject, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, prolonging survival, and / or improving a subject's physical or mental well-being. “Treatment,” “treating,” and “treat” include prophylactic treatment, prevention, and prevent, thereby slowing or preventing onset of disease, such as tuberculosis.

[0073] As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” refers to a detectable (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, such as an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased,” “reduced,” and the like encompass both a partial reduction and a complete reduction compared to a control.

[0074] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” or “enhanced” or “enhancing” or “enhance” refers to a detectable (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%) positive change in the parameter from a comparison control, such as an established normal or reference level of the parameter, or an established standard control.11B. Mutant Mycobacterium tuberculosis compositions

[0075] New and effective vaccines are urgently required against TB. Live-attenuated Mtb represent a class of TB vaccine candidates that can elicit highly protective responses, but there remain concerns about the safety of such strains.

[0076] The present disclosure provides nucleic acid construct comprising a Mycobacterium tuberculosis genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene. The present disclosure also provides a mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene. A “mutation” of a nucleic acid sequence (such as a gene) refers to any change in the nucleic acid sequence (such as the gene). Nonlimiting examples comprise insertions, deletions, duplications, substitutions, inversions, and translocations of any nucleic acid sequence, regardless of how the mutation is brought about and regardless of how or whether the mutation alters the functions or interactions of the nucleic acid. For example and without limitation, a mutation may result in the production of proteins with altered amino acid sequences (e.g. missense mutations, nonsense mutations, frameshift mutations, etc.) and / or the production of proteins with the same amino acid sequence (e.g. silent mutations). Certain synonymous mutations may create no observed change in a bacterium while others that encode for an identical protein sequence nevertheless result in an altered phenotype (e.g. due to codon usage bias, altered secondary protein structures, etc.). Mutations may occur within coding regions (e.g., open reading frames) or outside of coding regions (e.g., within promoters, terminators, untranslated elements, or enhancers), and may affect, for example and without limitation, gene expression levels, gene expression profiles, protein sequences, and / or sequences encoding RNA elements such as tRNAs, ribozymes, ribosome components, and microRNAs.

[0077] In specific embodiments, the present disclosure provides a nucleic acid construct containing a Mycobacterium tuberculosis genome containing a deletion of the sigH gene (ΔsigH) and a deletion of at least one additional gene, and a mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome comprising a deletion of the sigH gene (ΔsigH) and a deletion of at least one additional gene. A “deletion” of a gene as used herein refers to a deletion of one or more nucleic acids that comprises the gene. A deletion of a gene refers to both a partial deletion of the gene, and a complete deletion of the gene.12

[0078] The Mycobacterium tuberculosis deletion strains may carry a deletion of the sigH gene (ΔsigH) such as a complete deletion of the sigH gene. The sigH gene encodes for a transcription factor that, when induced, protects Mtb from the stress of lung macrophages and allows it to survive and cause disease. ΔsigH renders the infectious agent non-pathogenic and also protective against further Mtb challenge. “Non-pathogenic” as used herein refers to being unable to cause disease, such as tuberculosis. Thus, a ΔsigH Mycobacterium tuberculosis strain can elicit immune response against Mycobacterium tuberculosis to protect against further Mtb challenge, while not causing tuberculosis, including active, symptomatic, or infectious tuberculosis. In monkeys, pulmonary delivery of the ΔsigH strain significantly protects the monkeys from Mtb challenge and development of tuberculosis. An Mtb mutant ΔsigH exhibits remarkable efficacy as a vaccine in animal models by eliciting lung-specific T cell central memory (TCM) responses.

[0079] Throughout its life cycle in the host, Mtb encounters many stress conditions, the response to which is regulated by SigH, playing a key role in maintaining its viability. The ΔsigH mutant is attenuated ex vivo, as well as in macaques, where it induces strong lung immune signatures that protect against lethal TB. Although simian immunodeficiency virus (SIV) co-infection does not reactivate infection with the MtbΔsigH in macaques, additional unrelated mutations are required to ensure its clinical safety. For example, World Health Organizational (WHO) guidelines require that additional independent mutations be incorporated into ΔsigH prior to clinical testing. The Mycobacterium tuberculosis deletion strains provided herein includes at least one additional gene deletion to ΔsigH, and thus meet the Geneva consensus safety requirements, which state that any Mtb-based vaccine must include at least two independent gene deletions before being tested in human trials. The mutant Mycobacterium tuberculosis strains provided herein can be double knock-out (an additional gene deletion in addition to ΔsigH, DKO) or triple knock-out (two additional gene deletions in addition to ΔsigH, TKO) mutants.

[0080] Provided herein are MtbΔsigH-based TB vaccines and vaccine candidates that meet the Geneva consensus recommendations for safe live TB vaccines with strong potential for clinical advancement, deletion in which the ability of Mtb colonize macaque lungs is attenuated. To achieve this, eight specific genes to be deleted in the ΔsigH strain were identified. The selected genes and strains were: (i) ΔfbpAΔsapM, (ii) ΔsecA2ΔsodA, (iii) ΔleuD, (iv) ΔmetA, (v) Δmce4EΔmce4F, (vi) ΔhadC, (vii) ΔMT3785, and (viii) Δmce1A. The proteins encoded by the Mtb genes, fbpA and sapM interfere with phagolysosomal maturation in host and therefore, the Mtb ΔfbpAΔsapM strain has been shown to be significantly attenuated in murine and human macrophages due to increased13phagolysosomal 108 fusion that facilitates an increased presentation of Ag85B to CD4+ T-cells. This mutant is immunogenic and protective in macrophages and mice. Similarly, SecA2 facilitates the arrest of phagolysosomal fusion enhancing Mtb survival in host and transports proteins involved in pathogenesis, such as SodA, SapM, and PknG. As expected, Mtb ΔsecA2ΔsodA is highly attenuated and exhibits enhanced antigen presentation and phagolysosomal fusion capability. The MtbΔsecA2 mutant is also efficacious as a TB vaccine and safe in SIV co-infected macaques. Mtb ΔleuD and ΔmetA are attenuated auxotrophs. ΔleuDΔpanCD and ΔmetA strains of Mtb are safe in SCID mice, guinea pigs, immuno-competent and SIV co-infected macaques and exhibit protection against Mtb challenge. The remaining genes were selected because transposon interruption in these alleles rendered Mtb avirulent in macaques and in various other screening studies involving in silico, macrophage or murine models. None of the products encoded by these genes are related to sigH -function or -signaling. Deletion mutants of several of these genes have been individually shown to be attenuated due to dysregulation in cholesterol (mce4) or mycolic acid (mce1) transport.

[0081] The strains provided herein can be generated using high-throughput gene-replacement strategies. For example, mutant Mycobacterium tuberculosis encoded by nucleic acid constructs comprising a Mycobacterium tuberculosis genome comprising a deletion of the sigH gene (ΔsigH) and a deletion of at least one additional gene may be generated using homologous recombination enabled by phage-mediated specialized transduction. Genes that can be deleted in the Mycobacterium tuberculosis strains provided herein include genes reported to generate immune enhancement- or auxotrophy-based attenuation phenotypes, or genes that render Mtb avirulent in macaque lungs. For example, the deletion of at least one gene in addition to ΔsigH can include a deletion of fbpA (ΔfbpA); a deletion of sapM (ΔsapM); a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM); a deletion of mce4E (Δmce4E); a deletion of mce4F (Δmce4F); a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F), a deletion of secA2 (ΔsecA2); a deletion of sodA (ΔsodA); a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA); a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC)); a deletion of Rv3683 (ΔRv3683); a deletion of leuD (ΔleuD); a deletion of panCD (ΔpanCD); a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD); a deletion of metA (ΔmetA); a deletion of cobM (ΔcobM); a deletion of mce1A (Δmce1A); a deletion of fadD29 (ΔfadD29); or a combination of any thereof..

[0082] The mutant or deletion strains of Mtb can be constructed for example using temperature-sensitive mycobacteriophage phAE159 and a shuttle cosmid vector pYUB854. Briefly, ~800bp upstream and downstream flanking regions of sigH are cloned into a modified pYUB854 cosmid, flanking an apramycin resistance gene. The recombinant cosmid is subsequently packaged into phage14DNA phAE159. The recombinant phagemid is electroporated in M. smegmatis to generate temperature sensitive mycobacteriophages. These phages are propagated in M. smegmatis at 30°C to obtain high-titer phage lysates and finally used to transduce Mtb at the non-permissive temperature of 37°C. Transductants are selected on 7H11 plates supplemented with apramycin. For generation of double and triple knockout (DKO / TKO) mutant strains devoid of two and three genes, the second and third target genes are replaced with a hygromycin and a kanamycin resistance gene, respectively, in the genome of MtbΔsigH. Deletion of genes in the Mtb genome is confirmed by PCR using locus specific primers and sequencing.

[0083] The premise of this approach is that all strains are by themselves attenuated and the addition of ΔsigH renders them more immunogenic. sigH controls the expression of anti-oxidants, which in turn protects Mtb from host oxidative burst and inhibits antigen presentation. Safety studies in healthy and SIV co-infected immunocompromised rhesus macaques can be conducted to evaluate the safety of the mutant Mycobacterium tuberculosis strains provided herein. In some embodiments, Mycobacterium tuberculosis strains provided herein are safe and nonpathogenic.

[0084] To further test the safety / immunogenicity / efficacy of the DKO / TKO mutant strains, appropriate animal models (such as SCID mice, SIV co-infected rhesus monkeys) can be used to down-select two strains. Two safest and most effective mutant strains can be extensively tested in a preclinical trial in rhesus monkeys involving a relevant low-dose, high-virulence Mtb challenge. Next, the top two strains can be combined with a mucosal delivery approach designed to optimally induce lung-specific responses. Any strain which works in these pre-clinical models to provide protection against Mtb challenge is ready to move to human clinical trials.

[0085] In specific embodiments, MtbΔsigH-based double and triple knockout (DKO and TKO) strains, collectively referred to herein as Mtb KOs, result in a safe and non-pathogenic infection in mammals, including humans, rhesus macaques, and cynomolgus macaques, even under conditions of SIV co-infection. The Mtb KOs provided herein, each incorporating the ΔsigH deletion alongside additional gene deletions targeting virulence, immune evasion, or metabolic pathways, exhibit strong attenuation, fail to disseminate to extrapulmonary organs, and induce robust antigen-specific multifunctional T cell responses in the airways. Notably, the challenge elicits inducible bronchus-associated lymphoid tissue (iBALT) formation, a correlate of protection, and significantly reduces pulmonary pathology compared to wild-type Mtb or Mtb / SIV co-infection controls. In specific embodiments, MtbΔsigHΔmce1A replicates in the lungs, yet does not cause disease, suggesting that the ΔsigH deletion mitigates the hypervirulence associated with Δmce1A. MtbΔsigH-based KOs15provided herein can be next-generation live attenuated TB vaccine candidates suitable for further clinical development.

[0086] Also provided is an immunogenic composition containing the mutant Mycobacterium tuberculosis provided herein. The immunogenic composition can contain a carrier or an excipient in addition to the active ingredient, the mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome containing a mutation (such as a deletion) of the sigH gene (ΔsigH) and a mutation (such as a deletion) of at least one additional gene.C. Preventing and Treating tuberculosis

[0087] A method of stimulating an immune response against Mycobacterium tuberculosis in a subject is provided. The method includes administering an effective amount of the mutant Mycobacterium tuberculosis or the immunogenic composition provided herein.

[0088] A method of treating tuberculosis in a subject is also provided. The method includes administering an effective amount of an immunogenic composition containing a mutant Mycobacterium tuberculosis or the immunogenic composition provided herein.

[0089] In some embodiments, the subject has or is at risk of developing tuberculosis and other comorbidity, such as human immunodeficiency virus co-infection (TB / HIV), and the method provided herein can treat co-infection, such as TB / HIV co-infection.

[0090] In the methods provided herein, the mutant Mycobacterium tuberculosis is encoded by a Mycobacterium tuberculosis genome containing a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene. In some embodiments, the mutant Mycobacterium tuberculosis is non-pathogenic.

[0091] In some embodiments, the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

[0092] The mutation in the sigH gene can be a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene can be a deletion of the at least one additional gene.

[0093] The deletion of at least one additional gene may include a deletion of fbpA (ΔfbpA); a deletion of sapM (ΔsapM); a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM); a deletion of mce4E (Δmce4E); a deletion of mce4F (Δmce4F); a deletion of mce4E (Δmce4E) and a deletion of mce4F16(Δmce4F), a deletion of secA2 (ΔsecA2); a deletion of sodA (ΔsodA); a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA); a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC)); a deletion of Rv3683 (ΔRv3683); a deletion of leuD (ΔleuD); a deletion of panCD (ΔpanCD); a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD); a deletion of metA (ΔmetA); a deletion of cobM (ΔcobM); a deletion of mce1A (Δmce1A); a deletion of fadD29 (ΔfadD29); or a combination of any thereof. The DKO and TKO mutant strains provided herein can have attenuated effects in both immunocompetent and SIV-co-infected macaques. Combinatorial infection with the DKO or TKO mutant strain can generate strong cellular immune responses in the lung, akin to MtbΔsigH. Aerosol infection with the DKO or TKO strains provided herein can elicit inducible Bronchus Associated Lymphoid Tissue (iBALT), which is a correlate of protection from TB. The structures and function of the DKO and TKO strains based on MtbΔsigH are further described in Arora et al. JCI Insight 2025 Aug 28:e195947. doi: 10.1172 / jci.insight.195947, the entire content of which is incorporated herein by reference.

[0094] In various instances, the immunogenic compositions described herein (for example, immunogenic composition comprising mutant Mycobacterium tuberculosis of the present disclosure) can be formulated for delivery to a subject via any route of administration known to a person of skill in the art. Modes of administration are commonly known or are apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co.1985).

[0095] Mutant Mycobacterium tuberculosis or immunogenic compositions comprising mutant Mycobacterium tuberculosis provided herein can delivered to a subject by any suitable route, including but not limited to for instance auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric,17occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated. In specific embodiments, mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis is delivered parenterally (such as intravenously, intraarterially, intranasally, intratracheally, intrabronchially, by inhalation, intramuscularly, subcutaneously, intradermally, topically, intraperitoneally, intrathecally). In specific embodiments, mutant Mycobacterium tuberculosis or immunogenic compositions comprising mutant Mycobacterium tuberculosis provided herein is delivered to a subject by intramuscular or subcutaneous administration.

[0096] In some embodiments, mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis is formulated for systemic administration. In some embodiments, mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis is formulated for delivery to a target organ, for example, to the lung.

[0097] An injectable composition for parenteral administration (for example intravenous, intramuscular, intrathecal intracerebrospinal fluid, or intranasal), can contain mutant Mycobacterium tuberculosis and optionally additional components in a suitable i.v. solution, such as sterile physiological salt solution. In other embodiments, the composition is formulated as a suspension in an aqueous emulsion.

[0098] Liquid immunogenic compositions can be prepared by dissolving or dispersing a population of mutant Mycobacterium tuberculosis or the binding agent-therapeutic agent conjugates described herein, and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension.

[0099] Intravenous formulations can comprise mutant Mycobacterium tuberculosis, an isotonic medium and one or more substances. Example intravenous or other parenteral fluid formulations may contain saline solutions (for example normal saline (NS); about 0.91% w / v of NaCl, about 300 mOsm / L) and / or dextrose 4% in 0.18% saline, and optionally 1%, 2% or 3% human serum albumin.18

[0100] For use in an oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers. In the case of intranasal, intratracheal or intrapulmonary administration, the compositions may be provided in a liquid formulation which can be sprayed into the nose, trachea and / or lungs.

[0101] When the composition is employed in the form of a solid preparation for oral administration, the preparation may be a tablet, granule, powder, capsule or the like. In a tablet formulation, the composition is typically formulated with additives, for example an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.

[0102] Mutant Mycobacterium tuberculosis or the immunogenic compositions comprising mutant Mycobacterium tuberculosis provided herein may be administered once to the subject or, alternatively, multiple administrations may be performed over a period of time (for example booster administration). For example, two, three, four, five, or more administrations may be given to the subject or over a set period of time. The duration of therapies can be a duration sufficient to have a therapeutic effect, such as normalization of elevated serum cytokine levels.

[0103] In some embodiments, mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis described herein is administered in one dose. In other embodiments, mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis described herein is administered in two or more doses. For example, the mutant Mycobacterium tuberculosis of the mutant Mycobacterium tuberculosis composition can be administered, in some embodiments, once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 8 weeks, or once every 12 weeks. In some embodiments, the number, frequency, or amount of subsequent doses is dependent on the achievement of a desired therapeutic effect. In some embodiments, the composition is administered to a subject at the frequency and amount required to achieve a therapeutic effect. In some embodiments, the subject can be monitored for desired therapeutic effects and unwanted side effects associated with administration of the composition.19

[0104] Mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis as described herein can be used in preventing or treating tuberculosis in a subject. The mutant can also prevent or treat tuberculosis and other comorbidity, such as human immunodeficiency virus co-infection (TB / HIV).

[0105] Mutant Mycobacterium tuberculosis or immunogenic compositions comprising mutant Mycobacterium tuberculosis as described herein may be administered to a subject as a monotherapy (a single agent) or in a combination therapy where the subject is administered an immunogenic composition comprising mutant Mycobacterium tuberculosis or the binding agent-therapeutic agent conjugates in combination with one or more additional agents. mutant Mycobacterium tuberculosis or an immunogenic composition comprising mutant Mycobacterium tuberculosis of the present disclosure, and one or more additional agents, can be administrated to a subject simultaneously, sequentially or temporally.

[0106] In some embodiments, administration of the therapeutically effective amount of mutant Mycobacterium tuberculosis prevents onset of tuberculosis or reduces symptoms of tuberculosis if infected. Administration of the therapeutically effective amount of mutant Mycobacterium tuberculosis can reduce morbidity or mortality associated with tuberculosis.

[0107] The following examples are provided to illustrate, but not to limit, the various embodiments of the compositions and methods described in this disclosure.EXAMPLESExample 1: Development of MtbΔsigH

[0108] FIG. 1 schematically depicts the genomic organization of pYUB854 cosmid and subsequent packaging strategy. Briefly, approximately 800bp upstream and downstream flanking regions of sigH were cloned into a modified cosmid vector (pYUB854) flanking an apramycin resistance gene. The subsequent cosmid containing sigH and flanking sequence were packaged into phage DNA (phAE159). The subsequent sigH phagemid was electroporated in M. smegmatis generating temperature sensitive mycobacteriophages. The phages were propagated in M. smegmatis at 30°C to generate high-titer phage lysates, which were ultimately used to transduce Mycobacterium tuberculosis (such as Mycobacterium tuberculosis CDC1551) at 37°C. Transduced Mycobacterium tuberculosis were grown and selected on 7H11 plates containing apramycin to ensure successful transduction.20Example 2: In vivo efficacy of MtbΔsigH vaccination in TB infected rhesus macaques

[0109] To test in the in vivo efficacy of MtbΔsigH vaccine in a large animal model, rhesus macaques were either unvaccinated or vaccinated with the standard of care Bacillus Calmette-Guérin (BCG) vaccine or with the MtbΔsigH vaccine followed by infection with TB. As shown in FIGs.2A and 2B indicate that MtbΔsigH vaccination displayed heightened efficacy over the standard of care BCG vaccine in conferring protection against TB. Compared to unvaccinated and BCG vaccinated rhesus macaques, MtbΔsigH vaccination led to a significantly decreased number of colony forming units (CFU) in the lungs and bronchial lymph nodes (LN) (FIGs.2A and 2B respectively), signifying a lower infection burden in these subjects. In line with these observations, FIG. 2C depicts immunofluorescent staining of lung tissue from MtbΔsigH vaccinated rhesus macaques post TB infection, which displays more CD20 staining compared to unvaccinated or BCG vaccinated subjects post TB infection. This indicates that MtbΔsigH vaccination enhances pulmonary B cell infiltration, potentially signifying a heightened level of immunity.Example 3: In vivo efficacy of MtbΔsigH vaccination in TB infected cynomolgus macaques

[0110] FIG. 3A-3C depicts results from in vivo efficacy testing of the MtbΔsigH vaccine compared to BCG vaccine in TB infected cynomolgus macaques, a more resistant model than rhesus macaques. Compared to unvaccinated or BCG vaccinated TB infected cynomolgus macaques, MtbΔsigH vaccination significantly lowered the CFUs per gram of lung (FIG.3A) and also increased the proportion of sterile lung lobes in the subjects (FIG.3B). Gross pathological lung features of TB infected cynomolgus macaques that were either unvaccinated or vaccinated with BCG or MtbΔsigH are displayed in FIG 3C, which demonstrates MtbΔsigH vaccination attenuates the pathological features of TB and decreases tuberculosis lesions in the lung.Example 4: Construction and in vitro characterization of ΔsigH-based double and triple knockout strains of M. tuberculosis

[0111] To comply with the Geneva consensus, live vaccine candidates must undergo multiple stable gene deletions in virulent Mtb. To adhere to the Geneva consensus safety requirement, a list of candidate genes for stable knockout in addition to ΔsigH was generated for both double knockout (DKO) and triple knockout (TKO) vaccines. The candidate genes were selected based on previous studies. Additionally, candidate knockout genes are not downstream of the sigH signaling pathway.21FIG. 4A provides a list of example vaccine candidates containing DKO and TKO Mycobacterium tuberculosis.

[0112] Constructed were the isogenic single mutants, ΔsapM::hygR, Δmce1A::hygR, ΔsecA2::hygR, ΔhadC::hygR, ΔMT3785::hygR, ΔmetA::hygR, ΔleuD::hygR, ΔfbpA::kanR as well as the double mutant, Δmce4E / Δmce4F::hygr in Mtb CDC1551 (FIGs. 4B1-4J2) using a specialized-transducing phage-based system. The entire open reading frame (ORF) of each gene was replaced by either a hygromycin (hygR) or kanamycin (kanR) resistance gene via homologous recombination using the fragments immediately up- and down-stream of the coding sequence (FIGs. 4B1-4J2). The deletions of secA2 and mce1A involved the removal of hygR selectable markers using γδ resolvase via phage transduction, resulting in unmarked mutants, MtbΔsecA2-un and MtbΔmce1A-un (FIGs.4G1-4H2). The sigH deletion was attained in the background of these single and double mutants by transduction with the recombinant phage phAE87-ΔsigH::apraR, a phagemid DNA carrying the upstream and downstream regions flanking sigH on either side of apraR cassette in a cosmid, pYUB854. sigH was then deleted by replacing the gene with an apramycin resistance cassette in each individually generated knockout strains of Mtb (FIGs.4J1-4J2). The deletions and replacements were confirmed by PCR analysis using either the external primer set flanking the gene or internal locus-specific primers or a mixture of both (FIGs.4B1-4J2). The results were also verified by sequencing. These observations confirmed that the open reading frames for the genes, sapM, fbpA, mce4E, mce4F, mce1A, secA2, hadC, MT3785, metA 142 and leuD, have been individually deleted in the MtbΔsigH strain.

[0113] As an initial step in assessing the contribution of each gene to bacterial fitness, the growth of the mutants was monitored in liquid cultures. The growth curves of the double (MtbΔ sigHΔ secA2, MtbΔ sigHΔ mce1A and MtbΔ sigHΔ MT3785) and triple knockouts (MtbΔ sigHΔ fbpAΔ sapM and MtbΔ sigHΔ mce4EΔ mce4F) were comparable to parental Mtb, with each strain reaching a similar plateau value with similar growth rates. MtbΔ sigHΔ metA and MtbΔ sigHΔ leuD also exhibited growth patterns similar to the wild type upon supplementation with methionine and leucine, respectively, consistent with previous findings for these auxotrophic mutants of Mtb (40, 41). MtbΔ sigHΔ hadC grew at a slightly slower rate compared to wild type and other strains. The colony morphology of all the mutant strains was also comparable to that of wild type Mtb. Each strain produced dry rough colonies characterized by ridges and well-defined borders. As reported earlier for MtbΔ hadC, compared to wild type and all other strains, MtbΔ sigHΔ hadC strain exhibited a revival-defective phenotype and a longer lag phase. In addition, the colony morphology of MtbΔ sigHΔ hadC22was noticeably different, with changes in texture and diminished pigmentation relative to the wild type, consistent with the previous findings for the inactivation of hadC in Mtb genome. Biofilm formation by Mtb facilitates its survival within the host and confers increased drug tolerance, while also hindering immune cell activity and evading host defenses. Strikingly, all mutant strains were severely compromised in their ability to form biofilm in vitro, including the isogenic mutant for MtbΔ sigH. To test whether the mutations introduced altered the drug susceptibility of parental strain, the MICs of Mtb KOs strains were measured to most frequently used first- and one second-line antitubercular drugs. All the mutant strains were sensitive to Rifampin (RIF), Isoniazid (INH) and Ethambutol (ETH). The MIC99 values of RIF against the mutant strains were <4 μM. The values fall between 0.39 μM to 0.78 μM for INH and 1.56 μM – 1683.12 μM for ETH.Example 5: Generation of MtbΔmce1AΔsigH DKO

[0114] FIG. 5A depicts the genomic organization of the mce1A locus and primers utilized for downstream PCR amplification. Through phage mediated deletion-substitution described in Example 1, the mce1A gene was deleted and replaced with hygromycin and sacB resistance sequences. FIG.5B depicts the PCR amplification products using primers flanking the mce1A locus in WT and KO strains. WT strains display a PCR product around 1.5kb indicative of the endogenous mce1A gene product. Δmce1A on the other hand display a PCR product ~4.0kb, indicating successful deletion of mce1A and insertion of the hygromycin resistance sequence. Insertion of the γΔ sequence was confirmed utilizing primers hygF and HR-R (FIG.5C), signifying successful transduction in the Δmce1A strain. An additional phage mediated unmarking step was performed to remove the hygromycin resistance sequence highlighted in FIG.5A. Using primers to amplify the hygromycin resistance cassette in the Δmce1A strain revealed PCR products in the Δmce1A strains that were marked with the hygromycin sequence but no products in the unmarked Δmce1A strain as shown in FIG. 5D. Using HR primers outlined in FIG. 5A, the γΔ sequence was confirmed to still be incorporated in the unmarked Δmce1A strain as evidenced by a larger PCR product of ~2.0kb compared to the ~750bp product observed in the WT strain. As shown in FIG.5E, the PCR product corresponds to the size of WT sigH and sigH KO (2.2 kb in KO; 855 bp in WT), indicating integration of the ^^^^ sequences into the genome and sigH gene deletion in the mce1 knockout-unmarked strain.Example 6: MtbΔsigH-based KOs challenge reduces in vivo bacterial burdens and does not disseminate to extra-thoracic organs23

[0115] Rhesus macaques were challenged with Mtb KOs strains (a mixture of all eight MtbΔsigH-based knockouts shown in FIGs.4B1-4I3, with a targeted dose of 50-100 CFU of each strain) via aerosol route and subsequently with SIV intravenously, were euthanized at the indicated time-points and analyzed for bacterial burdens.The primary indicator of protection is the thorough quantification of Mtb burden at necropsy. CFUs in the BALs of animals decreased by 75% after three weeks of challenge and remained low even after the SIV challenge (FIG.6A). As shown in FIGs.6A and 6B, one out of three SIV challenged macaques had higher bacterial burden at the time of necropsy but remained free of the disease as indicated by clinical parameters. As shown in FIGs.6C and 6F, the lung and bronchial lymph node (BrLN) bacillary loads in Mtb KOs / SIV-infected animals were 3.8- and 3.4-log10, respectively, in comparison to Mtb / SIV infected animals where these were 4.6- and 5.2-log10. Despite the high bacterial burdens, we observed 6.0-fold fewer granulomas in lungs of Mtb KOs / SIV-infected animals compared to WT / SIV control-infected NHPs (FIG.6D). Mtb / HIV coinfection in humans is marked by widespread dissemination to extra-thoracic organs. Therefore, the bacterial loads in spleen, liver and kidneys were evaluated. Mtb / SIV–co-infected macaques exhibited significant extrapulmonary dissemination as evident by high bacterial burdens (2-4 log10 CFU) in spleen, liver and kidneys. In contrast, no bacilli were detected in any of the collected extrapulmonary tissues of Mtb KOs / SIV-infected macaques. These results demonstrate MtbΔsigH-based KOs-challenged macaques remain asymptomatic of tuberculous disease throughout the study despite active replication in the lung and when infected with a high dose of pathogenic SIV did not result in reactivation of TB disease.Example 7: MtbΔmce1AΔsigH is a replicating strain in the MKOs challenged macaques

[0116] The Mtb mce1 operon has been implicated in fatty acid uptake. Previous studies have 2shown that the Δmce1A mutant of Mtb Erdman exhibits increased growth in BALB / c mice, leading to earlier mortality compared to mice infected with the parental strain. PCR analysis was performed on lysates prepared from colonies isolated from the BALs, lungs and BrLN at necropsy. This analysis was expected to reveal an amplification size corresponding to the Δmce1A genotype. Approximately 20% of the total CFUs recovered from all animals, were screened and as anticipated, mutations were mapped to mce1A in 91% of the colonies obtained from lungs. PCR analysis of the remaining 9% of the total screened clones identified them as either MtbΔsigHΔsecA2 or MtbΔsigHΔmce4EΔmce4F mutants. Thus, MtbΔsigHΔmce1A strain accounted for 3.7-log10 CFU, while MtbΔsigHΔsecA2 and MtbΔsigHΔmce4EΔmce4F strains contributed 0.7- log10 CFU and 0.9- log10, respectively, to the total24lung bacterial load (FIGs.6F-6H). The remaining five mutant strains did not replicate in the lungs (FIGs.6I-6M). None of the Mtb KOs other than ΔsigHΔmce1A survived in BAL and BrLN as all the colonies recovered from these tissues carry the mce1A deletion. These results suggest that the mce1A deletion likely enabled the strain to dominate and replicate in the thoracic region, while the sigH deletion continued to provide protection to the animals.Example 7: Challenge with MtbΔsigH-based DKO or TKO leads to reduced pulmonary pathology

[0117] It has been previously shown that MtbΔsigH induced minimal pathology, when aerosol vaccinated in macaques despite SIV co-infection. The detailed necropsies provided herein also showed that the MtbKOs (a mixture of all eight MtbΔsigH-based knockouts shown in FIGs.4B1-4I3, with a targeted dose of 50-100 CFU of each strain) and SIV-challenged group exhibited substantially fewer pulmonary lesions and less TB-related pathology, as evident by the gross- and histo-pathological analysis as well as computed tomography (CT) (FIGs.7A-7E). CT was used to evaluate pulmonary pathology parameters in lungs which were notably reduced in Mtb KOs and Mtb KOs / SIV group animals compared to those in Mtb wild-type and SIV co-infected animals, respectively (FIGs.7A and 7B), suggesting that SIV-induced pathology was not exacerbated in Mtb KOs-challenged animals. As shown in FIGs.7C and 7D, decreased gross pathology was observed in lungs from Mtb KOs and Mtb KOs / SIV infected animals in comparison to the control groups, Mtb WT and Mtb / SIV infected macaques, respectively. The lung tissues from the control group animals exhibited heavy tissue involvement with numerous tubercles (FIGs.7C and 7D). In contrast, lungs from Mtb KOs and Mtb KOs / SIV infected animals exhibited reduced pathology with minimum involvement (FIGs.7C and 7D). In concordance, high resolution scanning images showed a greater number of granulomas in sections from wild type Mtb / SIV co-infected animals in comparison to sections from Mtb KOs / SIV co258 infected macaques (FIG.7E). In the histopathological analysis, lesser tissue damage and reduced granuloma formation were observed in sections from Mtb KOs and Mtb KOs / SIV co-infected group (FIG. 7E). As shown in FIG. 7F, severe granulomatous inflammation and loss of parenchymal space was observed in sections from Mtb / SIV infected group. The detailed analysis of tissue damage in H & E-stained sections revealed that the extent of lung affected by TB lesions encompassed an average of 2% in case of Mtb KOs / SIV group whereas it was ~31% in case of Mtb / SIV-challenged animals (FIG.7G). Collectively, these observations suggest that MtbΔsigH- based KOs are safe and non-pathogenic in a stringent NHP model of TB.Example 8: Inducible bronchus-associated lymphoid tissue (iBALT) induction in MtbΔsigH-based KOs challenged animals25

[0118] The observed granulomas in lungs from the Mtb KOs and SIV co-infected macaques lacked the characteristic well-organized structure and appear to be iBALTs. Thus, these lung sections were further analyzed and subjected to Immunohistochemistry (IHC) staining for CD20+B cells, CD3+T cells and CD 68+macrophage / dendritic cells followed by confocal imaging. As apparent in the analogous H & E-stained sections, multiple organized lymphoid aggregates comprising B cells, T cells, and macrophages / monocytes were observed, consistent with the formation of iBALT structures. (FIGs.7H and 7I). Moreover, the IHC staining revealed that the observed granulomas in the lungs of Mtb KOs only and Mtb KOs / SIV co-infected macaques were surrounded by well-organized iBALT structures per lesion (data not shown). As shown in FIGs.7H and 7I, these follicles are predominantly composed of CD20⁺ B cells, surrounded by spherical layers of CD3+T cells reinforcing previous findings that activated B cells are crucial for the control of TB in macaques. Example 9: MtbΔsigH -based KOs challenge induces antigen specific T cell responses in airways

[0119] Since Mtb is an intracellular pathogen with pulmonary pathology driven by IFNγ, we next assessed whether the vaccination with MtbΔsigH -based KOs imparts protection against M. tuberculosis. It was shown that MtbΔsigH is immunogenic in rhesus and cynomolgus macaques inducing robust Mtb antigen specific T cell responses in airways with peak responses observed at week 5 post intramucosal vaccination. Therefore, we also evaluated the antigen specific T cell responses in BAL cells (FIGs.8A-8L) and lungs isolated at week 4 post MtbΔsigH-based KOs and upon subsequent SIV challenge. As expected, intramucosal Mtb KOs challenge was found to be immunogenic in rhesus macaques inducing antigen specific multi-functional T cell responses in CD4+(FIGs.8A and 8B) and CD8+(FIGs.8C and 8D) compartments. Among the BAL CD4+cells, almost 25-30% were producing IFN-γ and TNF-α simultaneously in response to Mtb CDC1551 whole cell lysate (WCL) or Mtb CDC1551 Cell Wall fraction (CW) and 15% could produce IL-2 with IFN-γ and TNF-α simultaneously (FIGs. 8A and 8B). In the CD8+compartment, 2.5% of cells were responding to WCL and 7.5% to CW by producing IFN-γ and 296 TNF-α simultaneously (FIG.8C).1% of the BAL CD8+T cells were producing IL-2 with IFN- 297 γ and TNF-α simultaneously in response to WCL while a higher fraction of 2.5 % were responsive to CW (FIG.8D). As expected, multi-functional responses to both WCL and CW were similar in CD4+compartment while CD8 multifunctionality was higher in CW due to presence of CD8+dominant antigens like CFP10. In the CD4+compartment, 25% stained positive for IFN-γ, 30% for TNF-α, 18-20% for IL-2, 4% for IL-17 in response to WCL and CW. In the CD8+ compartment, 2.5% stained positive for IFN-γ, 5% for TNF-α, 1.5% for IL-2, 3031% for IL-17 in response to WCL while 7.5% stained positive for IFN-γ,2612% for TNF-α, 3% for 304 IL-2, 2.5% for IL-17 in response to CW. At week 8 post Mtb KOs challenge / week 4 post SIV challenge, significant CD4+ T cells depletion was observed as expected (FIG. 5G). However, the Mtb WCL specific responses including the multi-functional T cell responses were retained in CD8+ compartment (FIGs.8E and 8F). When compared to naïve and wild type Mtb challenged controls, superior antigen specific CD4+(FIGs.8G-8I) and CD8+(FIGs.8J-8L) T cell responses were induced by Mtb KOs challenge. In conclusion, challenge with pooled MtbΔsigH-based KOs elicited impressive protective multifunctional helper and cytotoxic T-cell responses in the airways urther strengthening their feasibility as a safe and effective vaccine against TB.

[0120] The foregoing description generally illustrates and describes various embodiments. It will, however, be understood by those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the technology as disclosed herein, and that it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as being illustrative, and not to be taken in a limiting sense. Accordingly, various features and characteristics as discussed herein may be selectively interchanged and applied to other illustrated and non-illustrated embodiments, and numerous variations, modifications, and additions further can be made thereto without departing from the spirit and scope of the embodiments as set forth in the appended claims.

[0121] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.27

Claims

CLAIMSWhat is claimed is:

1. A nucleic acid construct comprising a Mycobacterium tuberculosis genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene.

2. The nucleic acid construct of claim 1, wherein the mutation in the sigH gene is a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene is a deletion of the at least one additional gene.

3. The nucleic acid construct of claim 1 or 2, wherein the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

4. The nucleic acid construct of claim 2, wherein the deletion of the at least one additional gene comprises:a deletion of fbpA (ΔfbpA);a deletion of sapM (ΔsapM);a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM);a deletion of mce4E (Δmce4E);a deletion of mce4F (Δmce4F);a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F),a deletion of secA2 (ΔsecA2);a deletion of sodA (ΔsodA);a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA);a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC));a deletion of Rv3683 (ΔRv3683);a deletion of leuD (ΔleuD);a deletion of panCD (ΔpanCD);a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD);a deletion of metA (ΔmetA);a deletion of cobM (ΔcobM);a deletion of mce1A (Δmce1A);28a deletion of fadD29 (ΔfadD29);or a combination of any thereof.

5. A mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene.

6. The mutant Mycobacterium tuberculosis of claim 5, wherein the mutation in the sigH gene is a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene is a deletion of the at least one additional gene.

7. The mutant Mycobacterium tuberculosis of claim 5 or 6, wherein the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

8. The mutant Mycobacterium tuberculosis of claim 6, wherein the deletion of the at least one additional gene comprises:a deletion of fbpA (ΔfbpA);a deletion of sapM (ΔsapM);a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM);a deletion of mce4E (Δmce4E);a deletion of mce4F (Δmce4F);a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F),a deletion of secA2 (ΔsecA2);a deletion of sodA (ΔsodA);a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA);a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC));a deletion of Rv3683 (ΔRv3683);a deletion of leuD (ΔleuD);a deletion of panCD (ΔpanCD);a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD);a deletion of metA (ΔmetA);a deletion of cobM (ΔcobM);29a deletion of mce1A (Δmce1A);a deletion of fadD29 (ΔfadD29);or a combination of any thereof.

9. An immunogenic composition comprising the mutant Mycobacterium tuberculosis of any one of claims 5-8.

10. A method of stimulating an immune response against Mycobacterium tuberculosis in a subject, the method comprising administering an effective amount of the mutant Mycobacterium tuberculosis of any one of claims 5-8 or the immunogenic composition of claim 9.

11. The method of claim 10, comprising stimulating an immune response against Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infection.

12. A method of treating tuberculosis in a subject, the method comprising administering an effective amount of an immunogenic composition comprising a mutant Mycobacterium tuberculosis encoded by a Mycobacterium tuberculosis genome comprising a mutation in the sigH gene that reduces level or activity of the sigH gene and a mutation of at least one additional gene that reduces level or activity of the at least one additional gene.

13. The method of claim 12, wherein the mutation in the sigH gene is a deletion of the sigH gene (ΔsigH) and the mutation in the at least one additional gene is a deletion of the at least one additional gene.

14. The method of claim 12 or 13, wherein the at least one additional gene is selected from the group consisting of fbpA, sapM, mce4E, mce4F, secA2, sodA, Rv3683, leuD, panCD, metA, cobM, mce1A, fadD29, and Rv0637 (hadC).

15. The method of claim 13, wherein the deletion of the at least one additional gene comprises:a deletion of fbpA (ΔfbpA);a deletion of sapM (ΔsapM);a deletion of fbpA (ΔfbpA) and a deletion of sapM (ΔsapM);a deletion of mce4E (Δmce4E);a deletion of mce4F (Δmce4F);30a deletion of mce4E (Δmce4E) and a deletion of mce4F (Δmce4F),a deletion of secA2 (ΔsecA2);a deletion of sodA (ΔsodA);a deletion of secA2 (ΔsecA2) and a deletion of sodA (ΔsodA);a deletion of Rv0637 (hadC) (ΔRv0637 (ΔhadC));a deletion of Rv3683 (ΔRv3683);a deletion of leuD (ΔleuD);a deletion of panCD (ΔpanCD);a deletion of leuD (ΔleuD) and a deletion of panCD (ΔpanCD);a deletion of metA (ΔmetA);a deletion of cobM (ΔcobM);a deletion of mce1A (Δmce1A);a deletion of fadD29 (ΔfadD29);or a combination of any thereof.

16. The method of any one of claims 12-15, comprising treating Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infection in the subject.31