COMPOSITIONS AND METHODS FOR TREATING AN ACTIVE MYCOBACTERIUM TUBERCULOSIS INFECTION

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application serial nos. 61/679,612, filed August 3, 2012, and 61/791,213, filed March 15, 2013, all of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 712192000940SeqList.txt, date recorded: July 31, 2013 size: 36 KB).

BACKGROUND

Technical Field

[0003] The present disclosure relates to methods and compositions for treating a primary active M. tuberculosis infection or an active infection resulting from reactivation of a latent infection in a mammal and to methods and compositions for improving the efficacy of chemotherapy regimens against active M. tuberculosis infection.

Description of the Related Art

[0004] Tuberculosis (TB) is a chronic infectious disease caused by infection with

Mycobacterium tuberculosis and other Mycobacterium species. TB is a major pandemic disease in developing countries, as well as an increasing problem in developed areas of the world, claiming between 1.7 and 2 million lives annually. Although infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as an acute inflammation of the lungs, resulting in fever and a nonproductive cough. If untreated, serious complications and death typically result. The increase of multidrug- resistant TB (MDR-TB) further heightens this threat (Dye, Nat Rev Microbiol 2009;7:81-7).

[0005] The course of a . tuberculosis infection runs essentially through 3 phases. During the acute or active phase, the bacteria proliferate or actively multiply at an exponential, logarithmic, or semilogrithmic rate in the organs, until the immune response increases to the point at which it can control the infection whereupon the bacterial load peaks and starts declining. Although the mechanism is not fully understood it is believed that sensitized CD4+ T lymphocytes in concert with interferon gamma (IFN-gamma, γ-IFN) mediate control of the infection. Once the active immune response reduces the bacterial load and maintains it in check at a stable and low level, a latent phase is established. Previously, studies reported that during latency M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma. However, recent studies have demonstrated that even in latency, the stage of infection characterized by constant low bacterial numbers, at least part of the bacterial population remain in a state of active metabolism. (Talaat et al. 2007, J of Bact 189, 4265-74).

[0006] These bacteria therefore survive, maintain an active metabolism and minimally replicate in the face of a strong immune response. In the infected individual during latency there is therefore a balance between non-replicating bacteria (that may be very difficult for the immune system to detect as they are located intracellularly) and slowly replicating bacteria. In some cases, the latent infection enters reactivation, where the dormant bacteria start replicating again albeit at rates somewhat lower than the initial infection. It has been suggested that the transition of M. tuberculosis from primary infection to latency is accompanied by changes in gene expression (Honer zu Bentrup, 2001). It is also likely that changes in the antigen- specificity of the immune response occur, as the bacterium modulates gene expression during its transition from active replication to dormancy. The full nature of the immune response that controls latent infection and the factors that lead to reactivation are largely unknown. However, there is some evidence for a shift in the dominant cell types responsible. While CD4 T cells are essential and sufficient for control of infection during the acute phase, studies suggest that CD8 T cell responses are more important in the latent phase. Bacteria in this stage are typically not targeted by most of the preventive vaccines that are currently under development in the TB field as exemplified by the lack of activity when classical preventive vaccines are given to latently infected experimental animals (Turner et al. 2000 Infect Immun. 68:6:3674-9.).

[0007] Although TB can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. Current clinical practice for latent TB

(asymptomatic and non-contagious) is treatment with 6 to 9 months of isoniazid or other antibiotic or alternatively 4 months of rifampin. Active TB is treated with a combination of 4 medications for 6 to 8 weeks during which the majority of bacilli are thought to be killed, followed by two drugs for a total duration of 6 to 9 months. Duration of treatment depends on the number of doses given each week. In addition, although compliance with the treatment regimen is critical, patient behavior is difficult to monitor. Some patients do not complete the course of treatment either due to side effects or the extreme duration of treatment (6-9 months), which studies have shown can lead to ineffective treatment and the development of drug resistance.

[0008] In order to control the spread of tuberculosis, both effective prophylactic vaccination and accurate early diagnosis of active disease followed by more effective therapeutic regimes including therapeutic vaccines and cost effective and patient accepted chemotherapeutics is of utmost importance. Currently prophylactic vaccination with live bacteria such as Bacillus Calmette-Gueerin (BCG), an avirulent strain of M. bovis, is the most efficient method for inducing protective immunity. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public with this agent. The development of molecular adjuvants, combined with select recombinant proteins, has enabled the development of a new generation of vaccines that may be used prophylactically as well as therapeutically to treat, as well as prevent infectious diseases. See, e.g., EP 2457926. What is needed is a therapeutic vaccine that is effective in stimulating an immune response for active TB disease even in the face of high bacterial burden in order to provide an adjunct to chemotherapeutics to reduce the treatment time, clear bacilli, limit lung pathology associated with disease and potentially limit the spread of MDR- TB.

[0009] Thus, there is an urgent need for new more effective therapeutic regimens for active M. tuberculosis infections that increase treatment compliance by reducing the treatment time in order to decrease TB transmission.

BRIEF SUMMARY

[0010] The present disclosure relates to methods of treating an active M. tuberculosis infection or an active infection resulting from reactivation of a latent infection in a mammal and to methods of improving the efficacy of chemotherapy regimens against active M.

tuberculosis infection.

[0011] The present disclosure is based on the surprising discovery that an active M.

tuberculosis infection can be effectively treated by a treatment regime comprising a therapeutic Mtb composition such as a therapeutic Mtb vaccine and chemotherapeutic agent effective against a M. tuberculosis infection, thereby shortening the chemotherapy time required for protection, reducing bacterial burden, and/or extending survival. Further, surprisingly, the inventors have discovered that the therapeutic Mtb composition when delivered during an active TB infection as an adjunct to antibiotic therapy can produce a beneficial immune response to M. tuberculosis that improves the efficacy of a

chemotherapeutic regime to TB disease. The inventors further discovered that administration of a therapeutic Mtb composition such as a therapeutic vaccine during an active TB infection adjunctively with a chemotherapeutic agent effective against a M. tuberculosis infection stimulated a significantly more robust, high quality (polyfunctional), and durable T _H l-type CD4 ⁺ T cell response.

[0012] Therefore, in one aspect, there is provided a method for treating an active tuberculosis infection in a mammal, the method comprising the step of administering to a mammal having an active infection with tuberculosis (e.g., M. tuberculosis) a chemotherapy agent and an immunologically effective amount of a therapeutic vaccine wherein the vaccine comprises a pharmaceutical composition comprising an Mtb antigen or an immunogenic fragment thereof from a Mycobacterium species of the tuberculosis complex and an adjuvant.

[0013] It will be understood in this and related methods of the disclosure that at least one step of administering the therapeutic vaccine, typically the initial step of administering the therapeutic vaccine, will take place when the mammal is actively infected with M.

tuberculosis and/or exhibits at least one clinical symptom or positive assay result associated with active infection. It will also be understood that the methods of the present disclosure may further comprise additional steps of administering the same or another therapeutic vaccine of the present disclosure at one or more additional time points thereafter, irrespective of whether the active infection or symptoms thereof are still present in the mammal, and irrespective of whether an assay result associated with active infection is still positive, in order to improve the efficacy of chemotherapy regimens. It will also be understood that the methods of the present disclosure may include the administration of the therapeutic vaccine either alone or in conjunction with other agents and, as such, the therapeutic vaccine may be one of a plurality of treatment components as part of a broader therapeutic treatment regime. Accordingly, the methods of the present disclosure advantageously improve the efficacy of a chemotherapy treatment regime for the treatment of an active tuberculosis infection.

[0014] In certain embodiments, the therapeutic vaccine comprises an isolated fusion polypeptide comprising a combination of two or more covalently linked M. tuberculosis antigens, or immunogenic fragments thereof, wherein the antigens are selected from the group consisting of Rv0164, Rv0496, Rv2608, Rv3020, Rv3478, Rv3619, Rv3620, Rvl738, Rvl813, Rv3810, Rv2389, Rv2866, Rv3876, Rv0054, Rv0410, Rv0655, Rv0831, Rvl009, Rvl099, Rvl240, Rvl288, Rvl410, Rvl569, Rvl789, Rvl818, Rvl860, Rvl886, Rvl908, Rv2220, Rv2032, Rv2623, Rv2875, Rv3044, Rv3310, Rv3881, Rv0577, Rvl626, Rv0733, Rv2520, Rvl253, Rvl980, Rv3628, Rvl884, Rv3872, Rv3873, Rvl51 1 and Rv3875, and antigens having at least 90% identity to any of the foregoing sequences.

[0015] In a specific embodiment, the therapeutic vaccine comprises the ID93 fusion polypeptide, which comprises the antigens Rv2608, Rv3619, Rv3620 and Rvl813.

[0016] In another specific embodiment, the therapeutic vaccine comprises the ID93 fusion polypeptide, which comprises the antigens Rv2608, Rv3619, Rv3620 and Rvl813, wherein the sequences of the antigens are from M. tuberculosis. In a more specific embodiment, the ID93 fusion polypeptide comprises a sequence set forth in SEQ ID NO: 1, or a sequence having at least 90% identity thereto.

[0017] Also provided herein is a method for treating an active tuberculosis infection in a mammal, the method comprising the step of administering to a mammal having an active tuberculosis infection an immunologically effective amount of a therapeutic vaccine in conjunction with one or more chemotherapeutic agents, wherein the vaccine comprises a pharmaceutical composition comprising an isolated fusion polypeptide, wherein the fusion polypeptide comprises (a) a combination of antigen Rv3620, and Rv2608 from a

Mycobacterium species of a tuberculosis complex and the antigens are covalently linked, or (b) a sequence having at least 90% identity to the combination of antigens.

[0018] Also provided herein is a method for treating an active tuberculosis infection in a mammal, the method comprising the step of administering to a mammal having an active tuberculosis infection an immunologically effective amount of a therapeutic vaccine in conjunction with one or more chemotherapeutic agents, wherein the vaccine comprises a pharmaceutical composition comprising an isolated fusion polypeptide, wherein the fusion polypeptide comprises (a) a combination of antigen Rvl813, Rv3620, and Rv2608 from a Mycobacterium species of a tuberculosis complex and the antigens are covalently linked, or (b) a sequence having at least 90% identity to the combination of antigens.

[0019] In certain embodiments, the active infection to be treated according to the disclosed methods is an active infection that is causing a clinical symptom of active TB in the mammal, selected from the group consisting of weakness, fever, chills, weight loss, anorexia and night sweats. In other embodiments, the active infection is causing a clinical symptom of pulmonary TB symptoms in the mammal, selected from the group consisting of persistent cough, thick mucus, chest pain and hemoptysis. In still other embodiments, the active infection is characterized by Mtb bacteria which proliferate, reproduce, expand or actively multiply at an exponential, logarithmic, or semilogrithmic rate in an organ of the mammal. In other more specific embodiments, the active infection is identified using an assay selected from the group consisting of an acid fast staining (AFS) assay; a bacterial culture assay, such as the BACTEC MGIT 960 assay; an IGR test, such as the QFT®-Gold test or the QFT®- Gold In-tube T SPOT™.TB test; a skin test, such as the TST Mantoux skin test (TST); and intracellular cytokine staining of whole blood or isolated PBMC following antigen stimulation.

[0020] It will be apparent that, in some embodiments, the active infection will be an active primary infection of M. tuberculosis, while in others it will result from reactivation of a latent infection of M. tuberculosis. In some embodiments, the mammal will be infected with a multidrug resistant (MDR) strain of M. tuberculosis. In other embodiments, the mammal will have been previously immunized with Bacillus Calmette-Guerin (BCG).

[0021] Certain embodiments of the disclosed methods include administration of one or more chemotherapeutic agents effective in treating a M. tuberculosis infection, such as isoniazid and/or rifampin. In some situations, the mammal is first administered one or more

chemotherapeutic agents over a period of time and then administered the therapeutic vaccine. In other situations, the mammal is first administered the therapeutic vaccine and then administered one or more chemotherapeutic agents over a period of time. In still other situations, administration of the one or more chemotherapeutic agents and the therapeutic vaccine is initiated at the same time. Further still, it will be understood that when practicing the disclosed methods it may be desirable to administer the pharmaceutical composition and/or therapeutic vaccine to the mammal on multiple occasions, e.g., one or more subsequent times after the first administration.

[0022] In some embodiments, the therapeutic vaccine further comprises an adjuvant. In some embodiments, the adjuvant used in the therapeutic vaccine is a GLA adjuvant, such as a GLA adjuvant having the following structure:

wherein R ¹ , R ³ , R ⁵ and R ⁶ are C _U -C ₂ o alkyl; and R ² and R ⁴ are C ₁₂ -C ₂₀ alkyl or C ₉ -C ₂₀ alkyl.

[0023] In a more specific embodiment, when using a GLA having the above structure, R 1 , R 3 ,

R ⁵ and R ⁶ are C _11-14 alkyl; and R ² and R ⁴ are C _12-15 alkyl. In an even more specific

embodiment, the GLA of the above structure is one in which R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R 2 and R 4 are C ₁₃ alkyl. In an even more specific embodiment, the GLA of the above structure is one in which R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R ² and R ⁴ are C9 alkyl.

[0024] In another aspect, the compositions are employed in methods for reducing the time course of chemotherapy in an active tuberculosis infection in a subject, the method

comprising the step of administering to a mammal with an active Mycobacterium

tuberculosis infection an immunologically effective amount of a therapeutic vaccine as described herein, e.g., comprising a fusion protein or polypeptide or an immunogenic fragment thereof from a Mycobacterium species of the tuberculosis complex and an adjuvant, adjunctively with one or more chemotherapeutic agents effective against a M. tuberculosis infection, thereby reducing the time course of chemotherapy against a M. tuberculosis infection. In another aspect, provided herein is a method for reducing the time course of chemotherapy against an active tuberculosis infection, the method comprising administering to a mammal having an active tuberculosis infection an immunologically effective amount of a therapeutic vaccine in conjunction with the chemotherapy, wherein the vaccine comprises a pharmaceutical composition comprising an isolated fusion polypeptide, wherein the fusion polypeptide comprises (a) a combination of antigen Rvl813, Rv3620, and Rv2608 from a Mycobacterium species of a tuberculosis complex and the antigens are covalently linked, or (b) a sequence having at least 90% identity to the combination of antigens, and wherein the vaccine induces an immune response against tuberculosis, thereby providing for a reduced time course of the chemotherapy against an active tuberculosis infection. In some aspects, the time course of therapy is shortened to about 3, 4, 5, 6, or 7 months, e.g., no more than about 3, 4, 5, 6, or 7 months. By shortening the time course of chemotherapy against a M.

tuberculosis infection, the present methods are also effective in enhancing the compliance of an individual being treated for an active M. tuberculosis infection in completing an entire course of treatment.

[0025] In a further aspect, the compositions are employed in methods for stimulating a polyfunctional, durable T _H l-type CD4 ⁺ T cell response in an active tuberculosis infection in a subject, the method comprising the step of administering to a mammal with an active Mycobacterium tuberculosis infection an immunologically effective amount of a therapeutic vaccine as described herein, e.g., comprising a fusion protein or an immunogenic fragment thereof from a Mycobacterium species of the tuberculosis complex and an adjuvant, adjunctively with one or more chemotherapeutic agents effective against a M. tuberculosis infection, thereby stimulating a polyfunctional, durable Tnl-type CD4 ⁺ T cell response. By shortening the time course of chemotherapy against a M. tuberculosis infection, the present methods are also effective in enhancing the compliance of an individual being treated for an active M. tuberculosis infection in completing an entire course of treatment.

[0026] In any of the aforementioned embodiments, a mammal may be a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 shows the bacterial burden and survival of SWR/J and C57BL/6 mice infected with Mycobacterium tuberculosis (Mtb) and treated with antibiotics. SWR/J and C57BL/6 mice were infected with a low dose (50-100 bacteria) aerosol (LDA) of Mtb H37Rv (ATCC #27294). (FIG. 1A) The number of viable bacteria in the lungs (5 mice/group) were determined 15, 30 and 100 days after infection. Symbols indicate the mean +/- the standard deviation. (FIG. IB) The survival of SWR/J and C57BL/6 mice was monitored in animals (8 mice/group) infected with Mtb H37Rv and mock- or treated with a 90-day antibiotic regimen (Rx 90d) consisting of INH and RIF administered on days 30-120. (FIG. 1C) SWR/J mice were infected with a LDA of Mtb H37Rv and treated with 30, 60 or 90 days of antibiotics starting on day 15 (Rx 30d, 60d, 90d) or on day 30 (Rx 90d (30)). Survival of SWR/J mice (7 mice/group) is shown. (FIG. ID) Number of viable bacteria in the lungs of animals (5 mice/group) mock- or treated with a 90-day INH/RIF regimen (Rx 90d) administered on days 30-120, was determined 30, 60, 90, 120 and 150 days after infection. * P < 0.05 (1-way

ANOVA followed by Dunnett's Multiple Comparison Test or Logrank test) is considered significant. One representative of two experiments is shown.

[0029] Figure 2 shows the colony-forming unit counts and survival of SWR/J mice infected with a LDA of Mtb and treated with antibiotics and ID93/GLA-SE. SWR/J mice were infected with LDA of Mtb (day 0). Fifteen days later (day 15) mice were mock- or antibiotics-treated for 90 days (Rx 90d). A subset of antibiotic-treated mice in each group was also immunized 3x 3 weeks apart with ID93/GLA-SE either during (DTT; days 15, 36, 57) or post-antibiotic therapeutic treatment (PTT; days 107, 128, 149). (FIG. 2A) Scheme of immunotherapy experiments. (FIG. 2B) Number of viable bacteria in the lungs of animals (6 or 7 mice/group) was determined 177 days after infection. * P < 0.05 is considered significant. (FIG. 2C) Protection was assessed by monitoring animal deaths (9 or 10 mice/group) caused by Mtb over time. One representative of four experiments is shown. P < 0.05 (Logrank test) is considered significant.

[0030] Figure 3 shows survival of SWR/J Mice infected with Mtb and treated with the ID93/GLA-SE vaccine and reduced antibiotic chemotherapy. SWR/J mice were infected with a LDA of Mtb H37Rv. Fifteen days later mice were treated for 60 or 90 days with antibiotics (Rx 60d and Rx 90, respectively). Following the completion of the 60 day antibiotic regimen, mice were immunized 3x 3 weeks apart with ID93/GLA-SE. (FIG. 3A) Protection was assessed by monitoring animal deaths (7 mice/group) caused by Mtb over time. P < 0.05 (Logrank test) is considered significant. (FIGS. 3B-3M) Histopathological evaluation of lung tissues post-challenge with Mtb H37Rv. Inflammatory responses and granuloma (g) formation are shown in H&E sections (FIGS. 3B-3I) and the presence of AFB (arrows) (FIGS. 3J-3M) was evaluated. (FIGS. 3B, 3F, 3J) Mock-treated mice, day 106; (FIGS. 3C, 3J and 3K) 90-day antibiotic therapy, day 106; (FIGS. 3D, 3H, 3L) 90-day antibiotic therapy + ID93/GLA-SE, day 241; (FIGS. 3E, 31 and 3M) 60-day antibiotic therapy + ID93/GLA-SE, day 295 Data shown are representative of 5 mice/group. One representative of three experiments is shown.

[0031] Figure 4 shows ID93-specific cytokine responses in SWR mice following

immunotherapy. SWR mice were infected with a LDA Mtb H37Rv and treated with either 90 days of antibiotics alone or antibiotics followed by immunization with ID93/GLA-SE 3x 3 weeks apart. (FIG. 4A) Cytokine profile of ID93-stimulated splenocytes recovered at either day 177 or 241 post-infection. Cells were incubated for 24 hours in the presence of antigen or media control and supernatants were collected and analyzed by multiplex bead array for IFN- γ, IL-2, TNF, IL-5, IL-10, IL-13, and IL-17. Box plots show median and interquartile range after background subtraction. P-values from Wilcoxon rank sums test. (FIGS. 4B-4D) Intracellular cytokine staining for ID93-specific T-cell responses at days 149 and 177 postinfection. Cells were stimulated with ID93 or media control in the presence of brefeldin A for 8-12 hours, stained with fluorochromeconjugated antibodies against CD3, CD4, CD8, CD44, IFN-γ, IL-2 and TNF. (FIGS. 4B and 4C) The panels show the gating scheme for FACS analysis. (FIG. 4D) Box plots in lower panel show median and interquartile range after background subtraction. P-values from Wilcoxon rank sums test. One representative of two experiments is shown.

[0032] Figure 5 shows survival, clinical parameters and bacterial burden of Non-Human Primates (NHP) infected with Mtb and treated with antibiotics and ID 93/ GLA-SE.

Cynomolgus macaques were inoculated intratracheally with 1000 CFU of virulent M.

tuberculosis (Erdman strain). The infection was allowed to proceed for 60 days followed by treatment with 30 days of INH/RIF antibiotics delivered by gavage or saline (Mock).

Monkeys (7 per group) were injected with ID93/GLA-SE (Rx + ID93/GLA-SE) administered 3 times 2 weeks apart or did not receive further treatment (Mock, Rx). (FIG. 5 A) Scheme of NHP immunotherapy experiment. (FIG. 5B) Survival was monitored for 50 weeks post exposure. (FIG. 5C) CYR changes were also evaluated monthly for 50 weeks post exposure. (FIG. 5D) At necropsy bacteria were quantified by enumerating the bacteriological burden (CFU) in monkey lungs. (FIG. 5E) Histologic appearance of H&E-stained sections of lung tissues harvested from NHP.

[0033] Figure 6 shows lung log ₁₀ CFU counts after (FIG. 6A) 6 weeks and (FIG. 6B) 12 weeks of treatment.

[0034] Figure 7 shows bacterial growth following termination of therapy.

DETAILED DESCRIPTION

[0035] As described herein, the present disclosure relates generally to compositions and methods for treating active TB infection using therapeutic TB vaccines in combination with anti-TB chemotherapeutic agents, which may lead to shortened treatment times, clearance of TB bacilli, and potentially limiting the spread of MDR-TB.

[0036] The therapeutic vaccine compositions of the present invention generally comprise at least two heterologous polypeptides of a Mycobacterium species of the tuberculosis complex. A Mycobacterium species of the tuberculosis complex includes those species traditionally considered as causing the disease tuberculosis, as well as Mycobacterium environmental and opportunistic species that cause tuberculosis and lung disease in immune compromised patients, such as patients with AIDS, e.g., Mycobacterium tuberculosis (Mtb),

Mycobacterium bovis, or Mycobacterium africanum, BCG, Mycobacterium avium,

Mycobacterium intracellulare, Mycobacterium celatum, Mycobacterium genavense,

Mycobacterium haemophilum, Mycobacterium kansasii, Mycobacterium simiae,

Mycobacterium vaccae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum (see, e.g., Harrison's Principles of Internal Medicine, volume 1, pp. 1004-1014 and 1019-1020). In a preferred embodiment, the Mycobacterium species to be prevented, treated or diagnosed according to the invention is Mycobacterium tuberculosis (Mtb). The sequences of antigens from Mycobacterium species are readily available. For example, Mycobacterium tuberculosis sequences can be found in Cole et al., Nature 393:531 (1998) and can be found at websites such as those maintained by the Wellcome Trust, Sanger Institute and Institut Pasteur.

[0037] In certain embodiments, the therapeutic vaccine comprises a fusion polynucleotide, fusion polypeptide, or composition, as described in US Patent Application Publication No. 2010/0129391 (the content of which are specifically incorporated herein by reference in its entirety).

[0038] For example, in certain specific embodiments, the therapeutic vaccine comprises an isolated fusion polypeptide or protein, or a polynucleotide encoding the same, comprising a combination of two or more covalently linked Mycobacterium tuberculosis antigens, or immunogenic fragments thereof, wherein the antigens are selected from the group consisting of Rv0164, Rv0496, Rv2608, Rv3020, Rv3478, Rv3619, Rv3620, Rvl738, Rvl813, Rv3810, Rv2389, Rv2866, Rv3876, Rv0054, Rv0410, Rv0655, Rv0831, Rvl009, Rvl099, Rvl240, Rvl288, Rvl410, Rvl569, Rvl789, Rvl818, Rvl860, Rvl886, Rvl908, Rv2220, Rv2032, Rv2623, Rv2875, Rv3044, Rv3310, Rv3881, Rv0577, Rvl626, Rv0733, Rv2520, Rvl253, Rvl980, Rv3628, Rvl884, Rv3872, Rv3873, Rvl51 1 and Rv3875, and antigens having at least 90% identity to any of the foregoing sequences, as described in US Patent Application Publication No. 2010/0129391.

[0039] In some embodiments, the therapeutic vaccine comprises an isolated fusion polypeptide comprising (a) a combination of antigen Rv3620, and Rv2608 from a

Mycobacterium species of a tuberculosis complex and the antigens are covalently linked, or (b) a sequence having at least 90% identity to the combination of antigens. In some embodiments, the therapeutic vaccine comprises an isolated fusion polypeptide comprising (a) a combination of antigen Rvl813, Rv3620, and Rv2608 from a Mycobacterium species of a tuberculosis complex and the antigens are covalently linked, or (b) a sequence having at least 90% identity to the combination of antigens. In some embodiments, the therapeutic vaccine comprises a fusion polypeptide comprising a combination of Mycobacterium antigens Rv2608, Rv3619, Rv3620 and Rvl813, or a sequence having at least 90% identity to the combination of antigens. In some embodiments, the Mycobacterium antigens Rv2608, Rv3619, Rv3620 and Rvl813 are M. tuberculosis antigens Rv2608, Rv3619, Rv3620 and Rvl813. In some embodiments, the fusion polypeptide comprises a sequence set forth in SEQ ID NO: l, or a sequence having at least 90% identity thereto. In some embodiments, the fusion polypeptide comprises a sequence set forth in SEQ ID NO:2, or a sequence having at least 90% identity thereto. In some embodiments, the therapeutic vaccine comprises a fusion polypeptide comprising a combination of Mycobacterium antigens Rv2608, Rv3620 and Rvl813, or a sequence having at least 90% identity the combination of antigens. In some embodiments, the Mycobacterium antigens Rv2608, Rv3620 and Rvl813 are M. tuberculosis antigens Rv2608, Rv3620 and Rvl813. In some embodiments, the fusion polypeptide comprises a sequence set forth in SEQ ID NO:3 or 4, or a sequence having at least 90% identity to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, antigen Rvl813 comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, antigen Rv3620 comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, antigen Rv2608 comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, antigen Rv3619 comprises the amino acid sequence of SEQ ID NO: 8. One skilled in the art would understand that one or more N-terminal amino acids (such as signal sequences) may be removed.

[0040] In a more specific embodiment, the therapeutic vaccine comprises the ID93 fusion protein, or a polynucleotide encoding the same, which comprises four antigens belonging to families of Mtb proteins associated with virulence (Rv2608, Rv3619, Rv3620) or latency (Rvl813), as described in US Patent Application Publication No. 2010/0129391 (specifically incorporated herein by reference in its entirety).

[0041] In some specific further embodiments, a fusion protein, e.g., an ID93 fusion protein, is formulated as a vaccine. In further specific embodiments, a therapeutic vaccine comprises a stable oil-in-water emulsion (SE) and GLA a synthetic TLR-4 agonist (GLA) as described in US Patent Application Publication No. 2008/0131466 (specifically incorporated herein by reference in its entirety). As one of ordinary skill in the art will understand, in some embodiments the therapeutic vaccine comprises an isolated polypeptide, an isolated fusion polypeptide or fragment (e.g., an antigenic/immunogenic portion) from a Mycobacterium species of the tuberculosis complex known in the art. Mtb polypeptides of the disclosure, antigenic/immunogenic fragments thereof, and other variants may be prepared using conventional recombinant and/or synthetic techniques.

[0042] In some embodiments, a nucleic acid molecule or fusion protein is administered with one or more chemotherapeutic agents effective against a M. tuberculosis infection. Examples of such chemotherapeutic agents include, but are not limited to, amikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin,

pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones. Such chemotherapy is determined by the judgment of the treating physician using preferred drug combinations. "First-line" chemotherapeutic agents used to treat a M. tuberculosis infection that is not drug resistant include isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide. "Second- line" chemotherapeutic agents used to treat a M. tuberculosis infection that has demonstrated drug resistance to one or more "first-line" drugs and include but are not limited to ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin.

[0043] In some embodiments, a therapeutic vaccine is administered to a mammal with active TB before, concurrently with, or after administration of the one or more chemotherapeutic agents effective against a M. tuberculosis infection. In some embodiments the

chemotherapeutic is administered concurrently, at the same time. Alternatively, a

chemotherapeutic is administered within minutes such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 minutes, hours such as about 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or even days such as about 1, 2, 3, 4, 5, or 6 days. In some embodiments, a chemotherapeutic is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks before the therapeutic vaccine. In one embodiment, a nucleic acid molecule or fusion protein is administered about 2 weeks after commencing administration of one or more chemotherapeutic agents. The one or more chemotherapeutic agents are generally administered over a period of time, for example, for about 1, 2, 3, or 4 weeks, or about 2, 3, 4, 5, 6 or 8 months, or about 1 year or longer.

[0044] In some embodiments, a first administration in a mammal with an active TB infection of a therapeutic composition for stimulating an immune response comprising a nucleic acid molecule, fusion polypeptide, or vaccine is followed by one or more subsequent administrations of a nucleic acid, fusion polypeptide, or vaccine. For instance, a first administration with a nucleic acid molecule or fusion polypeptide is followed by one or more subsequent administrations of a nucleic acid molecule or fusion protein. In one embodiment, a first administration with a nucleic acid molecule or fusion polypeptide is followed by one or more subsequent administrations of a fusion polypeptide. In one embodiment, a first administration with a nucleic acid molecule or fusion polypeptide is followed by one or more subsequent administrations of a nucleic acid molecule. Usually the first or second or subsequent administrations are given about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart, or up to about 4, 5, or 6 months apart. Further administrations are given about 6 months apart, or as long as 1, 2, 3, 4 or 5 years apart.

[0045] In another aspect, the compositions are employed in methods for reducing or shortening the time course of chemotherapy against a M. tuberculosis infection, the method comprising administering to a mammal already infected with Mycobacterium tuberculosis one or more chemotherapeutic agents effective against a M. tuberculosis infection and an immunologically effective amount of a pharmaceutical composition comprising a fusion polypeptide, e.g., ID93, or an immunogenic fragment thereof from a Mycobacterium species of the tuberculosis complex and an adjuvant, wherein said ID93 fusion polypeptide induces an immune response against M. tuberculosis, thereby allowing for reducing or shortening the time course of chemotherapy against a M. tuberculosis infection. Usually, administration of a nucleic acid molecule, fusion polypeptide, or vaccine will allow effective chemotherapeutic treatment against a M. tuberculosis infection within 6 months, 5 months, 4 months, 3 months, or less.

[0046] The compositions and methods of the present disclosure are usually administered to humans, but are effective in other mammals including domestic mammals (i.e., dogs, cats, rabbits, rats, mice, guinea pigs, hamsters, chinchillas) and agricultural mammals (i.e., cows, pigs, sheep, goats, horses).

Definitions

[0047] In the present description, the terms "about" and "consisting essentially of mean + 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have" and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0048] A "chemotherapeutic", "chemotherapeutic agents" or "chemotherapy regime" is a drug or combination of drugs used to treat or in the treatment thereof of patients infected or exposed to any species of M. tuberculosis and includes, but is not limited to, amikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid (ΓΝΗ), kanamycin, pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones and other derivatives analogs or biosimilars in the art. "First-line" chemotherapeutic agents are chemotherapeutic agents used to treat an M. tuberculosis infection that is not drug resistant and include but are not limited isoniazid, rifampin, ethambutol, streptomycin and

pyrazinamide and other derivatives analogs or biosimilars in the art. "Second-line" chemotherapeutic agents used to treat a M. tuberculosis infection that has demonstrated drug resistance to one or more "first-line" drugs include without limitation ofloxacin,

ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin and other derivatives analogs or biosimilars in the art.

[0049] As used herein "improving the efficacy of chemotherapy regimens" refers to shortening the duration of therapy required to achieve a desirable clinical outcome, reducing the number of different chemotherapeutics required to achieve a desirable clinical outcome, reducing the dosage of chemotherapeutics required to achieve a desirable clinical outcome, decreasing the pathology of the host or host organs associated with an active clinical infection, improving the viability of the host or organs of a host treated by the methods, reducing the development or incidence of MDR-TB strains, and/or increasing patient compliance with chemotherapy regimens.

[0050] A "therapeutic Mtb composition(s)" as used herein refers to a composition(s) capable of eliciting a beneficial immune response to M. tuberculosis when administered to a host with an active TB infection. A "beneficial immune response" is one that lessens signs or symptoms of active TB disease, reduces bacillus counts, reduces pathology associated with active TB disease, elicits an appropriate cytokine profile associated with resolution of disease, expands antigen specific CD4 ⁺ and CD8 ⁺ T cells, or improves the efficacy of chemotherapy regimens. Therapeutic Mtb compositions of the disclosure include without limitation polynucleotides which encode polypeptides, polypeptides, antigenic fragments, peptides, delivered in pharmaceutically acceptable formulations by methods known in the art and include vaccine formulations.

[0051] A "host", "subject", "patient", "mammal", or "individual" are all used herein interchangeably.

[0052] " . tuberculosis ", "Mtb", "Mycobacterium tuberculosis", "bacteria ", "bacterium", "bacillus " as used herein all refer to the bacteria responsible for causing TB disease in a mammal.

[0053] A "drug resistant" M. tuberculosis infection refers to a M. tuberculosis infection wherein the infecting strain is not held static or killed (is resistant to) one or more of so-called "frontline" chemotherapeutic agents effective in treating a M. tuberculosis infection {e.g., isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide).

[0054] A "multi-drug resistant", "MDR-TB" M. tuberculosis infection refers to a M.

tuberculosis infection wherein the infecting strain is resistant to two or more of "front-line" chemotherapeutic agents effective in treating a M. tuberculosis infection. Multi-drug resistant M. tuberculosis infections as used herein also refer to "extensively drug-resistant

tuberculosis" ("XDR-TB") as defined by the World Health Global task Force in October 2006 as a multi-drug resistant TB with resistance to any one of the fluoroquinolones (FQs) and at least one of the injectable drugs such as kanamycin, amikacin, and capreomycin.

[0055] "Active Tuberculosis", "Active TB", "TB Disease", "TB" or "Active Infection" as used herein refers to an illness, condition, or state in a mammal (e.g., a primate such as a human) in which Mtb bacteria are actively multiplying and invading organs of the mammal and causing symptoms or about to cause signs, symptoms or other clinical manifestations, most commonly in the lungs (pulmonary active TB) or can be due to an initial infection of the host. Clinical symptoms of active TB may include weakness, fatigue, fever, chills, weight loss, loss of appetite, anorexia, or night sweats. Pulmonary active TB symptoms include cough persisting for several weeks (e.g., at least 3 weeks), thick mucus, chest pain, and hemoptysis. "Reactivation tuberculosis" as used herein refers to active TB that develops in an individual having LTBI and in whom activation of dormant foci of infection results in actively multiplying Mtb bacteria. "Actively multiplying" as used herein refers to Mtb bacteria which proliferate, reproduce, expand or actively multiply at an exponential, logarithmic, or semilogrithmic rate in the organs of an infected host. In certain embodiments, an infected mammal (e.g., human) has a suppressed immune system. The immune suppression may be due to age (e.g., very young or older) or due to other factors (e.g., substance abuse, organ transplant) or other conditions such as another infection (e.g., HIV infection), diabetes (e.g., diabetes mellitus), silicosis, head and neck cancer, leukemia, Hodgkin's disease, kidney disease, low body weight, corticosteroid treatment, or treatments for arthritis (e.g., rheumatoid arthritis) or Crohn's disease, or the like.

[0056] Tests for determining the presence of active TB or condition caused by actively multiplying Mtb bacteria are known in the art and include but are not limited to Acid Fast Staining (AFS) and direct microscopic examination of sputum, bronchoalveolar lavage, pleural effusion, tissue biopsy, cerebrospinal fluid effusion; bacterial culture such as the BACTEC MGIT 960 (Becton Dickinson, Franklin Lakes, NJ, USA); IGR tests including the QFT®-Gold, or QFT®-Gold In-tube T SPOT™.TB, skin testing such as the TST The Mantoux skin test (TST); and intracellular cytokine staining of whole blood or isolated PBMC following antigen stimulation.

[0057] "Latent Tuberculosis Infection", "LTBI", "Latentcy", or "Latent Disease", "Dormant Infection", as used herein refers to an infection with M. tuberculosis (MTB) that has been contained by the host immune system resulting in a dormancy which is characterized by constant low bacterial numbers but may also contain at least a part of the bacterial population which remains in a state of active metabolism including reproduction at a steady maintenance state. Latent TB infection is determined clinically by a positive TST or IGRA without signs, symptoms or radiographic evidence of active TB disease. Latently infected mammals are not "contagious" and cannot spread disease due to the very low bacterial counts associated with latent infections. Latent tuberculosis infection (LTBI) is treated with a medication or medications to kill the dormant bacteria. Treating LTBI greatly reduces the risk of the infection progressing to active tuberculosis (TB) later in life (i.e., it is given to prevent reactivation).

[0058] "Mycobacterium species of the tuberculosis complex" includes those species traditionally considered as causing the disease tuberculosis, as well as Mycobacterium environmental and opportunistic species that cause tuberculosis and lung disease in immune compromised patients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis, or M. africanum, BCG, M. avium, M. intracellulare, M. celatum, M. genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M. fortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005). [0059] "Progressive Primary Tuberculosis" as used herein refers to a TB Disease that develops within the first several years after initial exposure to and infection with Mtb, due to failure of the host immune system to adequately contain the initial infection.

[0060] A "method of treatment", as disclosed herein, refers generally to a method for treating an active tuberculosis infection in a mammal using a therapeutic vaccine in conjunction with a chemotherapeutic treatment regime. It will be understood in this and related methods of the disclosure that at least one step of administering the therapeutic vaccine, typically the initial step of administering the therapeutic vaccine, will take place when the mammal is actively infected with M. tuberculosis and/or exhibits at least one clinical symptom or positive assay result associated with active infection. It will also be understood that the methods of the present disclosure may further comprise additional steps of administering the same or another therapeutic vaccine of the present disclosure at one or more additional time points thereafter, irrespective of whether the active infection or symptoms thereof are still present in the mammal, and irrespective of whether an assay result associated with active infection is still positive, in order to improve the efficacy of chemotherapy regimens. It will also be understood that the methods of the present disclosure may include the administration of the therapeutic vaccine either alone or in conjunction with other agents and, as such, the therapeutic vaccine may be one of a plurality of treatment components as part of a broader therapeutic treatment regime. Accordingly, the methods of the present disclosure

advantageously improve the efficacy of a chemotherapy treatment regime for the treatment of an active tuberculosis infection.

Polypeptide Compositions

[0061] As noted, the present disclosure, in one aspect, provides isolated Mycobacterium polypeptides, as described herein, including fusion polypeptides, and compositions containing same, and their use in combination with chemotherapeutic agents for treating active TB infections. Generally, a polypeptide of the disclosure will be an isolated polypeptide and may be a fragment (e.g., an antigenic/immunogenic portion) from an amino acid sequence disclosed herein, or may comprise an entire amino acid sequence disclosed herein. Polypeptides of the disclosure, antigenic/immunogenic fragments thereof, and other variants may be prepared using conventional recombinant and/or synthetic techniques.

[0062] In certain embodiments, the polypeptides of the disclosure are

antigenic/immunogenic, i.e., they react detectably within an immunoassay (such as an ELISA or T cell stimulation assay) with antisera and/or T cells from an infected subject. Screening for immunogenic activity can be performed using techniques well known to the skilled artisan. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125 I-labeled Protein A.

[0063] As would be recognized by the skilled artisan, immunogenic portions of the polypeptides disclosed herein are also encompassed by the present disclosure. An

"immunogenic portion," as used herein, is a fragment of an immunogenic polypeptide of the disclosure that itself is immunologically reactive (i.e., specifically binds) with the B-cells and/or T cell surface antigen receptors that recognize the polypeptide. Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T cell lines or clones. As used herein, antisera and antibodies are "antigen- specific" if they specifically bind to an antigen (i.e., they react with the protein in an immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well-known techniques.

[0064] In a particular embodiment, an antigenic/immunogenic portion of a polypeptide of the present disclosure is a portion that reacts with antisera and/or T cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T cell reactivity assay). Preferably, the level of immunogenic activity of the

antigenic/immunogenic portion is at least about 50%, preferably at least about 70% and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide. In some instances, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having similar to or greater than about 100% or 150% or more immunogenic activity.

[0065] A polypeptide composition of the disclosure may also comprise one or more polypeptides that are immunologically reactive with T cells and/or antibodies generated against a polypeptide of the disclosure, particularly a polypeptide having an amino acid sequence disclosed herein, or to an immunogenic fragment or variant thereof. [0066] In another embodiment of the disclosure, polypeptides are provided that comprise one or more polypeptides that are capable of eliciting T cells and/or antibodies that are immunologically reactive with one or more polypeptides described herein, or one or more polypeptides encoded by contiguous polynucleotide sequences contained in the

polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or to one or more polynucleotide sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.

[0067] The present disclosure also provides polypeptide fragments, including

antigenic/immunogenic fragments, comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide composition set forth herein, or those encoded by a polynucleotide sequence set forth herein.

[0068] In another aspect, the present disclosure provides variants of the polypeptide compositions described herein. Polypeptide variants (e.g., any of antigens and fusion polypeptides described herein) generally encompassed by the present disclosure will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequence set forth herein.

[0069] A polypeptide "variant," as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the disclosure and evaluating their immunogenic activity as described herein using any of a number of techniques well known in the art.

[0070] For example, certain illustrative variants of the polypeptides of the disclosure include those in which one or more portions, such as an N-terminal leader sequence or

transmembrane domain, have been removed. Other illustrative variants include variants in which a small portion (e.g., about 1-30 amino acids) has been removed from the N- and/or C- terminal of a mature protein.

[0071] In many instances, a variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present disclosure and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics, e.g., with immunogenic characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, immunogenic variant or portion of a polypeptide of the disclosure, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table A.

[0072] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

TABLE A

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA cue CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG ecu

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

[0073] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982,

incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);

phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0074] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within 2 is preferred, those within .1 are particularly preferred, and those within .0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0075] As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 . 1); glutamate (+3.0 . 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 . 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (- 3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within 2 is preferred, those within .1 are particularly preferred, and those within .0.5 are even more particularly preferred.

[0076] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their

hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine;

glutamine and asparagine; and valine, leucine and isoleucine.

[0077] In addition, any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than

phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0078] Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

[0079] As noted above, polypeptides may comprise a signal (or leader) sequence at the N- terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide {e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0080] When comparing polypeptide sequences, two sequences are said to be "identical" if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0081] Optimal alignment of sequences for comparison may be conducted using the

Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA;

Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4: 11-17; Robinson, E.D. (197 1) Comb. Theor 77: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Nat'lAcad., Sci. USA 80:126- 730.

[0082] Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) /. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[0083] One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

[0084] In one preferred approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions {i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence {i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0085] In certain preferred embodiments of the disclosure, there are provided Mycobacterium tuberculosis fusion polypeptides, and polynucleotides encoding fusion polypeptides. Fusion polypeptide and fusion proteins refer to a polypeptide having at least two heterologous Mycobacterium sp. polypeptides, such as Mycobacterium tuberculosis polypeptides, covalently linked, either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein. Mycobacterium tuberculosis antigens are described in Cole et al., Nature 393:531 (1998), which discloses the entire Mycobacterium tuberculosis genome. Antigens from other Mycobacterium species that correspond to Mycobacterium tuberculosis antigens can be identified, e.g., using sequence comparison algorithms, as described herein, or other methods known to those of skill in the art, e.g., hybridization assays and antibody binding assays.

[0086] The fusion polypeptides of the disclosure generally comprise at least two antigenic polypeptides as described herein, and may further comprise other unrelated sequences, such as a sequence that assists in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or that assists in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

[0087] Fusion proteins may generally be prepared using standard techniques. Preferably, a fusion protein is expressed as a recombinant protein. For example, DNA sequences encoding the polypeptide components of a desired fusion may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

[0088] A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures, if desired. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Certain peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0089] The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

[0090] Within preferred embodiments, an immunological fusion partner for use in a fusion polypeptide of the disclosure is derived from protein D, a surface protein of the gram- negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g. , the first N-terminal 100 110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a lipoprotein D fusion partner is included on the N- terminus to provide the polypeptide with additional exogenous T cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non- structural protein from influenzae virus, NS 1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.

[0091] In another embodiment, an immunological fusion partner comprises an amino acid sequence derived from the protein known as LYTA, or a portion thereof (preferably a C- terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N- acetyl-Lalanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265- 292 (1986)). LYTA is an autolysin that specifically degrades certain bonds in the

peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 70:795-798 (1992)). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

[0092] In general, polypeptides and fusion polypeptides (as well as their encoding

polynucleotides) are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

Polynucleotide Compositions

[0093] The present disclosure, in another aspect, also provides isolated polynucleotides, particularly those encoding fusion polypeptides of this disclosure {e.g., ID93), as well as compositions comprising such polynucleotides. As used herein, the terms "DNA" and "polynucleotide" and "nucleic acid" refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a

polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms "DNA segment" and "polynucleotide" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

[0094] As will be understood by those skilled in the art, the polynucleotide sequences of this disclosure can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.

[0095] As will be recognized by the skilled artisan, polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Mycobacterium antigen or a portion thereof) or may comprise a variant, or a biological or antigenic functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the

immunogenicity of the encoded polypeptide is not diminished, relative to the native protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. The term "variants" also encompasses homologous genes of xenogenic origin.

[0096] In additional embodiments, the present disclosure provides isolated polynucleotides comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this disclosure that comprise at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that

"intermediate lengths", in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200 500; 500 1,000, and the like.

[0097] The polynucleotides of the present disclosure, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0098] Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present disclosure, for example polynucleotides that are optimized for human and/or primate codon selection.

Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

[0099] Mycobacterium polynucleotides and fusions thereof may be prepared, manipulated and/or expressed using any of a variety of well-established techniques known and available in the art.

[0100] For example, polynucleotide sequences or fragments thereof which encode polypeptides of the disclosure, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

[0101] As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

[0102] Moreover, the polynucleotide sequences of the present disclosure can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, expression and/or immunogenicity of the gene product.

[0103] In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989). A variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0104] The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

[0105] In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of (3-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will. [0106] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 753:516-544 (1987).

[0107] In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. (5:307-311 (1987)).

Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al., EMBO J. 3: 1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 77:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen- mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0108] An insect system may also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 97:3224-3227 (1994)).

[0109] In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus

transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 87:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. [0110] Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert.

Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20: 125-162 (1994)).

[0111] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and W138, which have specific cellular machinery and characteristic mechanisms for such post- translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

[0112] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1- 2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

[0113] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 77 :223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (ColbereGarapin et al., J. Mol. Biol. 750:1-14 (1981)); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers has gained popularity with such markers as anthocyanins, (3- glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55: 121-131 (1995)).

[0114] A variety of protocols for detecting and measuring the expression of polynucleotide- encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). These and other assays are described, among other places, in Hampton et al., Serological Methods, a

Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 758:1211-1216 (1983).

[0115] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemilumine scent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like. [0116] Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the disclosure may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.

[0117] In addition to recombinant production methods, polypeptides of the disclosure, and fragments thereof, may be produced by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 43 1 A Peptide Synthesizer (Perkin Elmer).

Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

Pharmaceutical and Vaccine Compositions

[0118] In another aspect, the present disclosure concerns formulations of one or more of the polynucleotide, polypeptide or other compositions disclosed herein in pharmaceutically- acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. Such pharmaceutical compositions are particularly preferred for use as vaccines when formulated with a suitable immunostimulant/adjuvant system. The compositions are also suitable for use in a diagnostic context.

[0119] It will also be understood that, if desired, the compositions of the disclosure may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically- active agents. There is virtually no limit to other components that may also be included, provided that the additional agents do not cause a significant adverse effect upon the objectives according to the disclosure.

[0120] In certain preferred embodiments the compositions of the disclosure are used as vaccines and are formulated in combination with one or more immuno stimulants. An immuno stimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in, for example, Powell & Newman, eds., Vaccine Design (the subunit and adjuvant approach) (1995).

[0121] Any of a variety of immunostimulants may be employed in the vaccines of this disclosure. For example, an adjuvant may be included. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A (natural or synthetic), Bortadella pertussis or Mycobacterium species or Mycobacterium derived proteins. Suitable adjuvants are commercially available as, for example, Freund' s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 and derivatives thereof (SmithKline Beecham,

Philadelphia, Pa.); CWS, TDM, Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides;

polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.

Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0122] Other illustrative adjuvants useful in the context of the disclosure include Toll-like receptor agonists, such as TLR7 agonists, TLR7/8 agonists, and the like. Still other illustrative adjuvants include imiquimod, gardiquimod, resiquimod, and related compounds.

[0123] Certain preferred vaccines employ adjuvant systems designed to induce an immune response predominantly of the Thl type. High levels of Thl-type cytokines (e.g., IFN-γ, TNF-αβ, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Thl- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Thl-type, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mossman & Coffman, Ann. Rev.

Immunol. 7: 145-173 (1989). [0124] Certain adjuvants for use in eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated

monophosphoryl lipid A (3D-MPLTM), together with an aluminum salt (U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034; and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immuno stimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352 (1996). Another illustrative adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharaiaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins. Other illustrative formulations include more than one saponin in the adjuvant combinations of the present disclosure, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, β-escin, or digitonin.

[0125] In other embodiments, the adjuvant is a glucopyranosyl lipid A (GLA) adjuvant, as described in U.S. Patent Application Publication No. 2008/0131466, the disclosure of which is incorporated herein by reference in its entirety. For example, in one embodiment, the GLA adjuvant used in the context of the present disclosure has the following structure:

wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn-Cio alk l; and R ² and R ⁴ are C ₁₂ -C ₂₀ alk l.

[0126] In a more specific embodiment, the GLA has the formula set forth above wherein R ¹ , R ³ , R ⁵ and R ⁶ are C _n -i4 alkyl; and R ² and R ⁴ are C ₁₂ _ ₁₅ alkyl.

[0127] In a more specific embodiment, the GLA has the formula set forth above wherein R ¹ , R ³ , R ⁵ and R ⁶ are C _n alkyl; and R ² and R ⁴ are C ₁₃ alkyl.

[0128] In some embodiments, the adjuvant is a GLA adjuvant (e.g., synthetic) having the following structure:

[0129] In certain embodiments of the above GLA structure, R ¹ , R ³ , R ⁵ and R ⁶ are Cn-C ₂ o alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In a more specific embodiment, R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R ² and R ⁴ are C ₁₃ alkyl. In another more specific embodiment, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₀ alkyl; and R ² and R ⁴ are alkyl. In certain embodiments of the above GLA structure, R ¹ , R ³ , R ⁵ and R ⁶ are Cn-C ₂ o alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In certain embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R ² and R ⁴ are C ₉ alkyl.

[0130] In certain embodiments, the adjuvant is a GLA adjuvant (e.g., synthetic) having the following structure:

[0131] In certain embodiments of the above GLA structure, R ¹ , R ³ , R ⁵ and R ⁶ are Cn-C ₂ o alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In certain embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R 2 and R 4 are C ₉ alkyl.

[0132] In certain embodiments, the adjuvant is a synthetic GLA adjuvant having the following structure:

[0133] In certain embodiments of the above GLA structure, R ¹ , R ³ , R ⁵ and R ⁶ are Cn-C ₂ o alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In certain embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R 2 and R 4 are C ₉ alkyl.

[0134] In certain embodiments, the adjuvant is a synthetic GLA adjuvant having the following structure:

[0135] In certain embodiments of the above GLA structure, R ¹ , R ³ , R ⁵ and R ⁶ are Cn-C ₂ o alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In certain embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R 2 and R 4 are C ₉ alkyl.

[0136] In certain embodiments, the adjuvant is a synthetic GLA adjuvant having the following structure:

[0137] In certain embodiments, the adjuvant is a synthetic GLA adjuvant having the following structure:

[0138] In certain embodiments, the adjuvant is a synthetic GLA adjuvant having the following structure:

[0139] In a particular embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D- MPLTM. adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other formulations comprise an oil-in- water emulsion and tocopherol. Another adjuvant formulation employing QS21, 3DMPLTM adjuvant and tocopherol in an oil-in- water emulsion is described in WO 95/17210.

[0140] Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative as disclosed in WO 00/09159. Other illustrative adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2, AS2\ AS2," SBAS-4, or SBAS6, available from SmithKline Beecham, Rixensart, Belgium), Detox, RC- 529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1. [0141] Compositions of the disclosure may also, or alternatively, comprise T cells specific for a Mycobacterium antigen. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient. Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.

[0142] T cells may be stimulated with a polypeptide of the disclosure, polynucleotide encoding such a polypeptide, and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide. Preferably, the polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.

[0143] T cells are considered to be specific for a polypeptide of the disclosure if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54: 1065-1070 (1994)). Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with a polypeptide of the disclosure (100ng/mll00μg/ml, preferably 200ng/ml-25μg/ml) for 3-7 days should result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1 (1998)). T cells that have been activated in response to a polypeptide, polynucleotide or polypeptide-expressing APC may be CD4+ and/or CD8+. Protein-specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion. [0144] In the pharmaceutical compositions of the disclosure, formulation of

pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.

[0145] In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to a subject. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be

incorporated directly with the food of the diet.

[0146] In certain circumstances it will be desirable to deliver the pharmaceutical

compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No.

5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as

hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0147] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0148] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human

administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

[0149] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0150] The compositions disclosed herein may be formulated in a neutral or salt form.

Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0151] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0152] The phrase "pharmaceutically- acceptable" refers to molecular entities and

compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

[0153] In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, polynucleotides, and peptide compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

[0154] In certain embodiments, the delivery may occur by use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the compositions of the present disclosure may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle or the like. The formulation and use of such delivery vehicles can be carried out using known and conventional techniques. Exemplar Embodiments

1. A method for treating an active tuberculosis infection in a mammal, the method comprising the step of administering to a mammal having an active tuberculosis infection a chemotherapy agent and an immunologically effective amount of a therapeutic vaccine, wherein the vaccine comprises a pharmaceutical composition comprising an Mtb antigen or an immunogenic fragment thereof from a Mycobacterium species of a tuberculosis complex.

2. The method of embodiment 1, wherein the therapeutic vaccine comprises an isolated fusion polypeptide comprising a combination of two or more covalently linked

Mycobacterium tuberculosis antigens, or immunogenic fragments thereof, wherein the antigens are selected from the group consisting of Rv0164, Rv0496, Rv2608, Rv3020, Rv3478, Rv3619, Rv3620, Rvl738, Rvl813, Rv3810, Rv2389, Rv2866, Rv3876, Rv0054, Rv0410, Rv0655, Rv0831, Rvl009, Rvl099, Rvl240, Rvl288, Rvl410, Rvl569, Rvl789, Rvl818, Rvl860, Rvl886, Rvl908, Rv2220, Rv2032, Rv2623, Rv2875, Rv3044, Rv3310, Rv3881, Rv0577, Rvl626, Rv0733, Rv2520, Rvl253, Rvl980, Rv3628, Rvl884, Rv3872, Rv3873, Rvl511 and Rv3875, and antigens having at least 90% identity to any of the foregoing sequences.

3. The method of embodiment 1, wherein the therapeutic vaccine comprises an ID93 fusion

polypeptide, wherein the ID93 fusion polypeptide comprises Mycobacterium antigens Rv2608, Rv3619, Rv3620 and Rvl813.

4. The method of embodiment 3, wherein the Mycobacterium antigens Rv2608, Rv3619,

Rv3620 and Rvl813 are M. tuberculosis antigens Rv2608, Rv3619, Rv3620 and Rvl813.

5. The method of embodiment 3, wherein the ID93 fusion polypeptide comprises a sequence set forth in SEQ ID NO: 1, or a sequence having at least 90% identity thereto.

6. The method of embodiment 1, wherein the active tuberculosis infection is associated with a clinical symptom of weakness, fatigue, fever, chills, weight loss, loss of appetite, anorexia, night sweats, or any combination thereof.

7. The method of embodiment 1, wherein the active tuberculosis infection is a pulmonary active TB infection. 8. The method of embodiment 7, wherein the pulmonary active tuberculosis infection is associated with a clinical symptom of persistent cough, thick mucus, chest pain, hemoptysis, or any combination thereof.

9. The method of embodiment 1, wherein the active tuberculosis infection is characterized by Mtb bacteria which proliferate, reproduce, expand or actively multiply at an exponential, logarithmic, or semilogrithmic rate in an organ of the mammal.

10. The method of embodiment 1, wherein the active tuberculosis infection is identified using an assay selected from the group consisting of an acid fast staining (AFS) assay; a bacterial culture assay, such as the BACTEC MGIT 960 assay; an IGR test, such as the QFT®-Gold test or the QFT®-Gold In-tube T SPOTTM.TB test; a skin test, such as the TST Mantoux skin test (TST); and intracellular cytokine staining of whole blood or isolated PBMC following antigen stimulation.

11. The method of embodiment 1, wherein the active tuberculosis infection is an active primary infection of M. tuberculosis.

12. The method of embodiment 1, wherein the active tuberculosis infection is a reactivation tuberculosis infection.

13. The method of embodiment 1, wherein the mammal is infected with a multidrug resistant (MDR) M. iwberculosis.

14. The method of embodiment 1, wherein the mammal was previously immunized with Bacillus Calmette-Guerin (BCG).

15. The method of embodiment 1, wherein the mammal is a human.

16. The method of embodiment 1, further comprising the administration of one or more chemotherapeutic agents effective in treating a M. tuberculosis infection.

17. The method of embodiment 16, wherein the one or more chemotherapeutic agents is isoniazid, rifampin, or a combination thereof.

18. The method of embodiment 16, wherein the mammal is first administered one or more chemotherapeutic agents over a period of time and subsequently administered the therapeutic vaccine.

19. The method of embodiment 16, wherein the mammal is first administered the therapeutic vaccine and subsequently administered one or more chemotherapeutic agents over a period of time.

20. The method of embodiment 16, wherein administration of the one or more chemotherapeutic agents and the therapeutic vaccine is concurrent. 21. The method of embodiment 1, further comprising administering the therapeutic vaccine to the mammal one or more subsequent times, wherein a tuberculosis infection remaining in the mammal at the one or more subsequent times may or may not be an active tuberculosis infection.

22. The method of embodiment 1, wherein the vaccine further comprises an adjuvant.

23. The method of embodiment 22, wherein the adjuvant is GLA, having the following structure:

wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn-Cio alk l; and R ² and R ⁴ are C12-C20 alkyl.

24. The method of embodiment 23, wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn-14 alkyl; and R ² and R ⁴ are C _12-15 alkyl.

25. The method of embodiment 23, wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R ² and R ⁴ are C13 alkyl.

26. A method for reducing the time course of chemotherapy against an active

tuberculosis infection, the method comprising administering to a mammal having an active tuberculosis infection one or more chemotherapeutic agents effective against M. tuberculosis and an immunologically effective amount of a therapeutic vaccine, where the vaccine comprises a pharmaceutical composition comprising a fusion polypeptide or an

immunogenic fragment thereof from a Mycobacterium species of a tuberculosis complex, and wherein the fusion polypeptide induces an immune response against M. tuberculosis, thereby providing for a reduced time course of chemotherapy against an active M.

tuberculosis infection.

27. The method of embodiment 26, wherein the fusion polypeptide comprises a combination of two or more covalently linked Mycobacterium tuberculosis antigens, or immunogenic fragments thereof, wherein the antigens are selected from the group consisting of Rv0164, Rv0496, Rv2608, Rv3020, Rv3478, Rv3619, Rv3620, Rvl738, Rvl813, Rv3810, Rv2389, Rv2866, Rv3876, Rv0054, Rv0410, Rv0655, Rv0831, Rvl009, Rvl099, Rvl240, Rvl288, Rvl410, Rvl569, Rvl789, Rvl818, Rvl860, Rvl886, Rvl908, Rv2220, Rv2032, Rv2623, Rv2875, Rv3044, Rv3310, Rv3881, Rv0577, Rvl626, Rv0733, Rv2520, Rvl253, Rvl980, Rv3628, Rvl884, Rv3872, Rv3873, Rvl511 and Rv3875, and antigens having at least 90% identity to any of the foregoing sequences.

28. The method of embodiment 27, wherein the fusion polypeptide comprises the ID93 fusion polypeptide, which comprises the antigens Rv2608, Rv3619, Rv3620 and Rvl813.

29. The method of embodiment 28, wherein the ID93 fusion polypeptide comprises a sequence set forth in SEQ ID NO: 1, or a sequence having at least 90% identity thereto.

30. The method of embodiment 26, wherein time course of chemotherapy is shortened to no more than about 3 months, about 5 months, or about 7 months.

31. The method of embodiment 26, wherein the vaccine further comprises an adjuvant.

32. The method of embodiment 31, wherein the adjuvant is GLA, having the following structure:

wherein R ¹ , R ³ , R ⁵ and R ⁶ are C _U -C ₂ o alkyl; and R ² and R ⁴ are C12-C20 alkyl.

33. The method of embodiment 32, wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn-14 alkyl; and R ² and R ⁴ are C _12-15 alkyl.

34. The method of embodiment 32, wherein R ¹ , R ³ , R ⁵ and R ⁶ are C11 alkyl; and R ² and R ⁴ are C13 alkyl.

35. A method for treating a patient diagnosed with an active tuberculosis infection, the method comprising administering to the patient a chemotherapy agent and an

immunologically effective amount of a therapeutic vaccine, wherein the vaccine comprises a pharmaceutical composition comprising an Mtb antigen or an immunogenic fragment thereof from a Mycobacterium species of a tuberculosis complex.

36. The method of embodiment 35, wherein the patient is human. 37. The method of embodiment 35, wherein the therapeutic vaccine comprises an isolated

fusion polypeptide comprising a combination of two or more covalently linked

Mycobacterium tuberculosis antigens, or immunogenic fragments thereof, wherein the antigens are selected from the group consisting of Rv0164, Rv0496, Rv2608, Rv3020, Rv3478, Rv3619, Rv3620, Rvl738, Rvl813, Rv3810, Rv2389, Rv2866, Rv3876, Rv0054, Rv0410, Rv0655, Rv0831, Rvl009, Rvl099, Rvl240, Rvl288, Rvl410, Rvl569, Rvl789, Rvl818, Rvl860, Rvl886, Rvl908, Rv2220, Rv2032, Rv2623, Rv2875, Rv3044, Rv3310, Rv3881, Rv0577, Rvl626, Rv0733, Rv2520, Rvl253, Rvl980, Rv3628, Rvl884, Rv3872, Rv3873, Rvl511 and Rv3875, and antigens having at least 90% identity to any of the foregoing sequences.

38. The method of embodiment 35, wherein the therapeutic vaccine comprises an ID93 fusion polypeptide, wherein the ID93 fusion polypeptide comprises Mycobacterium antigens Rv2608, Rv3619, Rv3620 and Rvl813.

39. The method of embodiment 38, wherein the Mycobacterium antigens Rv2608, Rv3619,

Rv3620 and Rvl813 are M. tuberculosis antigens Rv2608, Rv3619, Rv3620 and Rvl813.

40. The method of embodiment 38, wherein the ID93 fusion polypeptide comprises a sequence set forth in SEQ ID NO: 1, or a sequence having at least 90% identity thereto.

41. The method of embodiment 35, wherein the active tuberculosis infection is associated

with a clinical symptom of weakness, fatigue, fever, chills, weight loss, loss of appetite, anorexia, night sweats, or any combination thereof.

42. The method of claim 35, wherein the active tuberculosis infection is a pulmonary active TB infection.

43. The method of embodiment 42, wherein the pulmonary active tuberculosis infection is associated with a clinical symptom of persistent cough, thick mucus, chest pain, hemoptysis, or any combination thereof.

44. The method of embodiment 35, wherein the active tuberculosis infection is characterized by Mtb bacteria which proliferate, reproduce, expand or actively multiply at an exponential, logarithmic, or semilogrithmic rate in an organ of the patient.

45. The method of embodiment 35, wherein the active tuberculosis infection is identified using an assay selected from the group consisting of an acid fast staining (AFS) assay; a bacterial culture assay, such as the BACTEC MGIT 960 assay; an IGR test, such as the QFT®-Gold test or the QFT®-Gold In-tube T SPOTTM.TB test; a skin test, such as the TST Mantoux skin test (TST); and intracellular cytokine staining of whole blood or isolated PBMC following antigen stimulation.

46. The method of embodiment 35, wherein the active tuberculosis infection is an active primary infection of M. tuberculosis.

47. The method of embodiment 35, wherein the active tuberculosis infection is a reactivation tuberculosis infection.

48. The method of embodiment 35, wherein the patient is infected with a multidrug resistant (MDR) M. tuberculosis.

49. The method of embodiment 35, wherein the patient was previously immunized with Bacillus Calmette-Guerin (BCG).

50. The method of embodiment 35, wherein the patient is a mammal.

51. The method of embodiment 35, further comprising the administration of one or more chemotherapeutic agents effective in treating a M. tuberculosis infection.

52. The method of embodiment 51, wherein the one or more chemotherapeutic agents is isoniazid, rifampin, or a combination thereof.

53. The method of embodiment 51, wherein the patient is first administered one or more chemotherapeutic agents over a period of time and subsequently administered the therapeutic vaccine.

54. The method of embodiment 51, wherein the patient is first administered the therapeutic vaccine and subsequently administered one or more chemotherapeutic agents over a period of time.

55. The method of embodiment 51, wherein administration of the one or more chemotherapeutic agents and the therapeutic vaccine is concurrent.

56. The method of embodiment 35, further comprising administering the therapeutic vaccine to the patient one or more subsequent times, wherein a tuberculosis infection remaining in the patient at the one or more subsequent times may or may not be an active tuberculosis infection.

57. The method of embodiment 35, wherein the vaccine further comprises an adjuvant.

58. The method of embodiment 57, wherein the adjuvant is GLA, having the following structure: wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn o alk l; and R ² and R ⁴ are C12-C20 alkyl.

59. The method of embodiment 58, wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn-14 alkyl; and R ² and R ⁴ are C _12-15 alkyl.

60. The method of embodiment 58, wherein R ¹ , R ³ , R ⁵ and R ⁶ are Cn alkyl; and R ² and R ⁴ are C13 alkyl.

[0155] The following Examples are offered by way of illustration and not by way of limitation.

Example 1

Development of the SWR/J mouse model of TB relapse and reactivation of an active TB infection.

[0156] Female, age-matched (4-6 weeks) SWR/J and C57BL/6 mice were purchased from Jackson and Charles River Laboratories, respectively. Mice were infected with a low dose (50 - 100 bacteria) aerosol (LDA) of Mtb H37Rv (ATCC #35718) using a UW-Madison aerosol chamber. The number of bacilli present in mice with an active infection the number of viable bacteria in the lungs (5 mice/group) were determined 15, 30 and 100 days after infection by methods known in the art. Symbols indicate the mean +/- the standard deviation. SWR/J and C57BL/6 strains with an active TB infection, mice were infected with Mtb H37Rv as described (8 mice/group) and survival monitored. To access the effect of chemotherapeutics on the model, fifteen days post-infection, a subset of mice were started on a drug regimen of isoniazid (INH) (at 85 mg/L of drinking water) and rifampin (RIF) (at 50 mg/L of drinking water) administered for 30, 60 or 90 consecutive days (Rx 30d, Rx 60d, Rx 90d). An additional group of mice were started on a drug regimen of isoniazid (INH) (at 85 mg/L of drinking water) and rifampin (RIF) (at 50 mg/L of drinking water) (collectively herein referred to as chemotherapeutic treatment) at 30 days post infection administered for 90 consecutive days. Female mice are estimated to drink between 0.15 and 0.37 mL/g (Bachmanov AA etal, 2002) The minimum inhibitory concentrations for Mtb H37Rv are 0.25 μΜ for RIF and 1.0 μΜ for INH

[0157] In contrast to C57BL/6 mice (Russell,et. AL, 2010), and consistent with previous observations (Baldwin et al., J of Immunology 2012), SWR/J mice failed to transition to a chronic state after Mtb infection as indicated by increasing viral titers in the SWR/J compared to the C57BL/6 mice (Fig. 1A) and are representative of a model of active MTB infection. Mock-treated SWR/J mice succumbed to lethal Mtb infection with a median survival time (MST) of 116.5 days, while those treated with chemotherapeutics (RIF/INH) for 90 days had an MST of 247.5 days (P < 0.001 ; Logrank test) compared to mock or chemotherapeutic treated C57/BL/6 mice (Fig. IB).

[0158] To determine optimal length of chemotherapeutic treatment in the SWR/J mice, animals were treated with RIF/INH beginning on day 15 post infection for either 30, 60, or 90 days or an additional group that received chemotherapy beginning on day 30 post exposure for 90 days. Survival curves were monitored. Significant differences in survival and recoverable lung CFU between animals that were mock- or drug-treated for 30 (P < 0.0005; Logrank test), 60 (P < 0.05; Logrank test) or 90 days (P < 0.005; Logrank test) were observed (Fig. 1C). Changing the initiation of chemotherapy from 15 to 30 days post-infection did not significantly alter the long term efficacy of treatment (P > 0.50; Logrank test) (Fig. 1C). While 60 or 90 days of chemotherapy was sufficient to decrease the number of viable lung bacteria below the limit of detection (Fig. ID), these treatment regimens were insufficient to achieve clearance of Mtb in SWR/J mice.

Example 2

Evaluation of therapeutic efficacy of the TB vaccine ID93 + chemotherapy in the SWR/J mouse model of TB relapse and reactivation of an active TB infection.

[0159] TB vaccine fusion proteins JD83 and ID93 or their component antigens formulated with the TLR4 antagonist GLA-SE have been previously demonstrated to provide prophylactic protection against TB in mouse and guinea pig, models when administered in three doses (Baldwin, etal., 2009, Bertholet, etal., 2010). The ID93/GLA-SE vaccine was tested in the SWR/J model of active infection to determine if this formulation would provide immunotherapeutic benefit as measured by reduction of CFU or improved survival. SWR/J mice (6 or 7 mice per group) were infected with LDA of Mtb as described in Example 1. Fifteen days later (day 15) mice were mock- or antibiotics-treated for 90 days (Rx 90d). A subset of antibiotic-treated mice in each group were also immunized. Mice were immunized 3 times, 3 weeks apart with 8 μg of ID93protein formulated with 20 μg of GLA-SE either during (DTT; days 15, 36, 57) or post-antibiotic therapeutic treatment (PTT; days 107, 128, 149). Therapeutic efficacy was determined by tracking survival over time and by plating lung homogenates as previously described (Bertholet et.al., 2008). The ID93/GLA-SE vaccine administered therapeutically(- -) in the SWR/J mice model of active TB infection increased the frequency of survival after infection (P < 0.01).

[0160] Compared to chemotherapy (Rx) alone (-·-), immunization with the ID93/GLA-SE vaccine as an adjunct to chemotherapy (-■-) further reduced CFU by 0.643 loglO (P < 0.05) (Fig. 2B). No differences in lung CFU were observed between the groups administered GLA- SE adjuvant alone plus chemotherapy (Rx + GLA-SE (-H-)), compared to chemotherapy alone Rx (-·-) (P > 0.05) (Fig. 2B). Moreover, there was a significant difference between the post-exposure efficacy induced by the Rx+ID93/GLA-SE and the Rx+GLA-SE groups (4.419 + 0.17 vs. 4.938 + 0.16 loglO, P < 0.05), demonstrating that the adjunctive bactericidal effect observed in these studies is antigen dependent.

[0161] Administration of the vaccine as an immunotherapeutic adjunct to chemotherapy after (PTT) or during (DTT) 90 days of chemotherapy treatment prevented death in 52 % and 67 % of Mtb-infected mice, respectively, (P < 0.0001) (Fig. 2C).

Example 3

Administration of the therapeutic ID93/GLA-SE vaccine as an adjunct to chemotherapy reduces the duration of drug therapy required to prolong survival in an active TB infection.

[0162] Additional experiments were performed in the SWR/J mouse model of TB relapse and reactivation of an active TB infection to evaluate if administration of the therapeutic

ID93/GLASE vaccine could reduce the duration of drug therapy required to prolong survival in an active TB infection. SWR/J mice were infected with a LDA of Mtb H37Rv. Fifteen days later mice were treated for 60 or 90 days with antibiotics as previously described (Rx 60d and Rx 90, respectively). Following the completion of the 60 day antibiotic regimen, mice were immunized immunized 3 times, 3 weeks apart with 8 μg of ID93protein formulated with 20 μg of GLASE(Rx 60d + JD93/GLA-SE; days 77, 98, 119). Protection was assessed by monitoring animal deaths (7 mice/group) caused by Mtb over time (P < 0.05 (Logrank test) is considered significant). (B-M) Histopathological evaluation of lung tissues post-challenge with Mtb H37Rv. Inflammatory responses and granuloma (g) formation are shown in H&E sections (B-I) and the presence of AFB (arrows) (J-M) was evaluated. (B, F, J) Mock-treated mice, day 106; (C, J and K) 90-day antibiotic therapy, day 106; (D, H, L) 90- day antibiotic therapy + ID93/GLA-SE, day 241; (E, I and M) 60-day antibiotic therapy + ID93/GLA-SE, day 295. Data shown are representative of 5 mice/group

[0163] Whereas 40% of the animals receiving 90 days of chemotherapy alone (Rx90d; - A -) survived Mtb infection (MST 214 days), 100% of the animals receiving vaccine

immunotherapy after 60 days of chemotherapy (-·-) survived for at least 250 days (P < 0.05) (Fig. 3A). These studies demonstrate that vaccine immunotherapy could reduce the duration of drug therapy by at least 1/3 while preventing death for an extended period after chemotherapy was withdrawn.

[0164] In order to determine if antibiotics combined with ID93/GLA-SE reduced TB lung pathology, sections from mock-, Rx-, and Rx+ID93/GLA-SE-treated mice were taken for histological analysis (Histologic Findings are presented in Tablel (below) and Fig. 3B-M.

Table 1. Effects of ID93/GLA-SE immunotherapy on lung pathology of fb-infected SWR/J

Lesion Lung

Group Lung AFB ^C Granuloma Diagnosis

Grade (%) ^b

- Histiocytic alveolar and

-Coalescing interstitial pneumonia,

3-4

Mock macrophage nodules, moderate to marked;

Moderate- 40-100 6-30

(Day 106) with syncytial giant granulomatous lobar

Marked

cells bronchopneumonia.

- Numerous AFB in lesions

-No nodular

0-2 - Histiocytic alveolar and

Rx ^d granulomas,

Mild- interstitial pneumonia, mild to

0-40% <1 Few macrophages

(Day 106) Moderate moderate.

-Resolution of large

lesions - Minimal AFB in lesions

-No significant

- Histiocytic alveolar and

Rx ^d histiocytic

4 interstitial pneumonia, (Day 241, 41-100 <30 granulomas, no

Marked marked.

295) syncytial

- Many AFB in lesions macrophages

Rx ^d + - Histiocytic

2 - Histiocytic alveolar and ID93/ granulomas with

Mild- interstitial pneumonia, mild- GLA-SE ^f 11-40 <6 syncytial macrophages

Moderate moderate.

(Day 241) -Several small dense

- Few or no AFB in lesions lymphoid aggregates -Histiocytic

Rx ^e + granulomas with - Histiocytic alveolar and

1-3

ID93/ syncytial macrophages interstitial pneumonia,

Minimal- 0-40 <l-6

GLA-SE ^f -Minimal, multifocal, minimal-moderate,

Moderate

(Day 295) infiltration of . Few AFB in lesions

lymphocytes

"Data are representative of 3-5 animals per group

"Percent of lung tissue involved: Minimal (grade 1 or <10 ); Mild (grade 2 or 11-20%); Moderate

(grade 3 or 21-40%); Marked (grade 4 or 41-100%)

-Number of Acid Fast Bacteria (AFB) / High Power Field (HPF), 600x

"90 day INH/RIF chemotherapy initiated 15 days following infection with Mtb

e60 day INH/RIF chemotherapy initiated 15 days following infection with Mtb

'Mice were immunized 3 times, 3 wks apart after the administration of chemotherapy treatment.

[0165] The lungs of mock-treated mice had diffuse alveolar edema (Fig. 3B,F) with grade 3- 4 (40-100%) involvement of the lung parenchyma appearing greatly inflamed and necrotic as previously reported [29, 38], with numerous acid-fast bacilli (>30/600x high power field (HPF) (Fig. 3J). The lung sections of the chemotherapy alone (Rx90d) group showed obvious resolution of inflammatory lesions (Fig. 3 C,G) with only rare bacilli (<1/HPF) (Fig. 3 K; Table 1). At day 241, the lungs of Rx 90d+ID93/GLA-SE mice had numerous granulomas (Fig. D,H) and few bacilli (<6 organisms/HPF, 600x) (Fig. 3 L; Table 1). At day 295, lungs of mice treated with 60d antibiotics and immunized with ID93/GLA-SE showed no significant lesions (Fig. 3 E,I; Table 1) and few bacilli (Fig. 3 M).

[0166] The data demonstrates that the ID93/GLA-SE vaccine administered in conjunction with antibiotics could be used to shorten standard chemotherapy regimens in active TB infections (Fig. 3A).

Example 4

Immune responses in SWR/J mice receiving chemotherapy alone or chemotherapy plus ID93/GLA-SE vaccination.

Cytokine profile of ID93 -stimulated splenocytes

[0167] SWR/J mice were infected with a LDA Mtb H37Rv and treated with either 90 days of antibiotics alone or antibiotics followed by three immunizations with ID93/GLA-SE 3 weeks apart as described in Example 2. Cytokine profiles from supernatants of ID93-stimulated splenocytes (day 177 or 241 post-infection) were analyzed after incubation for 24 hours in the presence of antigen or media alone by multiplex bead array for IFN-γ, IL-2, TNF, IL-5, IL- 10, IL-13, and IL-17. Box plots show median and interquartile range after background subtraction. P-values from Wilcoxon rank sums test. Intracellular cytokine staining for ID93-specific T-cell responses at days 149 and 177 postinfection.

[0168] Cells were stimulated with ID93 or media control in the presence of brefeldin A for 8- 12 hours, stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8, CD44, IFN-γ, IL-2 and TNFand analyzed by FACS. (B and C) The panels show the gating scheme for FACS analysis. (D) Box plots in lower panel show median and interquartile range after background subtraction. P- values from Wilcoxon rank sums test. In response to in vitro re- stimulation with ID93, a subset of cytokines representing pro-inflammatory, as well as TH1 and TH2 functional groups, was significantly up-regulated (Fig. 4A). TNF, a soluble mediator of Mtb- specific immunity in infected individuals, was significantly up-regulated at day 241 in the group immunized with ID93/GLA-SE (P < 0.05). In addition, ID93-specific IFN-γ, IL-2, and IL-17 responses were detected, which were significantly higher in vaccinated animals compared to unvaccinated animals. No significant difference in the concentration of the TH2-type IL-5 cytokine was detected but significant ID93-specific IL-10 and IL-13 responses were measured at day 241.

[0169] Polyfunctional CD4+ TH1 cells have recently been described as a correlate of protection against Leishmania major, and have been implicated in limiting disease progression in human TB [39, 40]. Frequencies of CD4+ and CD8+ T cells producing IFN-γ, IL-2 and TNF were thus examined to determine the phenotype of ID93- specific T-cell responses (Fig. 4B-D; S2B). Higher frequencies of ID93-specific polyfunctional triple- positive and IFN-y+TNF+ double-positive CD4+ T cells were observed in mice receiving adjunctive immunotherapy compared to mice receiving only chemotherapy (P < 0.05), (Fig. 4B-D). High background responses of ID93- specific TNF in both the CD4+ and CD8+ T- cell subsets were observed which was likely due to the increased immune activation of an ongoing Mtb infection in these animals. Although ID93- specific responses in CD8+ T cells were lower in magnitude than those observed in the CD4+ compartment, there were significantly higher frequencies of double (IFN-y+TNF-i-) and triple positive (IFN-y+IL- 2+TNF+) CD8+ T cells in mice receiving adjunctive ID93/GLA-SE vaccination. Altogether, these data show that though there are many antigens present after Mtb infection that could be potentially primed and boosted continuously, ID93/GLA-SE administered adjunctively with antibiotics was successful at stimulating a significantly more robust, high-quality

(polyfunctional) and durable THl-type anti-ID93 CD4+ T-cell response.

Example 5 ID93/GLA-SE as an adjunct to antibiotic treatment in cynomolgus macaques.

[0170] In order to demonstrate the safety of ID93/GLA-SE when administered as an adjunct to antibiotics in NHP, macaques were administered three doses of the vaccine after one month of RIF/INH antibiotics (Fig. 5A). Injection- site reactions were minimal, with no more than barely perceptible erythema and edema (Draize scale range 0 - 1), and there were no significant changes in body weight and temperature (data not shown). All 7 (100%) of the Rx + ID93/GLA-SEimmunized NHP survived to the last time point evaluated, whereas 6 NHP (85.7%) in the antibiotics alone group and 3 NHP (42.8%, P=0.44) in the mock treated group survived to this point (Fig. 5B). Four monkeys treated with Rx+ID93/GLA-SE either had no radiological changes or resolved the Mtb infection before the end of the experiment (as evidenced by lung infiltrates on previously positive chest X-rays), whereas none of the macaques receiving Rx alone or mock treatments resolved their Mtb infection and remained chest X-ray positive (Fig. 5C). Forty percent of the macaques treated with Rx+ID93/GLA-SE responded dramatically to adjunctive immunotherapy by showing quantitative differences in Mtb bacterial numbers when compared to the Rx alone group; (P<0.05), (Fig. 5D).

Interestingly, the Rx+ID93/GLA-SE macaques that had lower CFU counts also had negative chest X-rays at the end of the experiment. There was also a correlation by histopathology between group assignment and the presence of disease tissue, with animals receiving

ID93/GLA-SE containing the most healthy organs and the saline group having the most diseased organs (p=0.003) (Fig. 5E). Overall, these results demonstrated that an ID93/GLA- SE vaccine was well tolerated as a post exposure immunotherapeutic agent in cynomolgus macaques.

Example 6

Immune responses in BALB/c mice receiving chemotherapy alone or chemotherapy plus

ID83/GLA-SE vaccination.

[0171] Six-week-old female BALB/c mice (Charles River, Wilmington, MA) were infected with M. tuberculosis H37Rv, using the Inhalation Exposure System (Glas-Col, Terre Haute,

IN) and a log phase broth culture (optical density at 600 nm of 1.0) diluted 10 in 7H9 broth with the goal of implanting 2.5-3.0 loglO CFU in the lungs. M. tuberculosis H37Rv was prepared from mouse-passaged, frozen in aliquots, and sub-cultured in Middlebrook 7H9 broth with 10% oleic acid-albumin-dextrose-catalase (OADC) (Fisher, Pittsburgh, PA) and

0.05% Tween 80 prior to infection. Five mice were sacrificed 1 day after infection to confirm the number of bacteria implanted. The remaining mice were randomized to the treatment groups indicated in Table 2. Treatment with rifampin (R), isoniazid (H) and pyrazinamide (Z) collectively RHZ started 26 days after infection on Day 0. Rifampin and isoniazid (Sigma, St. Louis, MO) were dissolved separately in distilled water at 1 mg/ml to produce the dosing solution. Pyrazinamide (Fisher Scientific, Suwanee, GA) was dissolved in distilled water at 15 mg/ml to produce the dosing solution. Solutions were prepared and aliquotted weekly and kept at 4°C prior to use. Four control groups received drug vehicle (water) + GLA-SE adjuvant, drug vehicle + ID83 vaccine, RHZ + vaccine vehicle (saline), and RHZ + GLA-SE adjuvant. The test group received RHZ + ID83 vaccine. RHZ and drug vehicle were administered 5 days per week, by gavage, for 12 weeks. R or vehicle was administered at least 1 hour before HZ or vehicle to avoid previously described

pharmacokinetic drug-drug interactions which limit R absorption.

[0172] ID83 was formulated as a stable oil-in- water emulsion adjuvanted with GLA (GLA- SE) by mixing 4 ml of vaccine mixture were prepared with 2 ml GLA-SE (0.040 mg/ml, 4% Oil), 0.1 ml ID83 (0.2 mg/ml) and 1.9 ml saline (NaCl). The vaccine preparation was vortexed briefly before use. 100 μΐ of vaccine was administered subcutaneously. The injected doses were: 2 μg of GLA-SE +/- 0.5 μg of ID83.

[0173] Vaccination of infected mice began 6 weeks after infection (2 weeks after treatment RHZ initiation). Three doses of vaccine or controls (saline or adjuvant only) were administered subcutaneously in 100 microliters at 3 week intervals (i.e., after 2, 5 and 8 weeks of treatment). The vaccine contained 2 micrograms of GLA-SE adjuvant and 0.5 micrograms of ID83. Controls included saline only and adjuvant only. The site of injection was rotated.

Table 2. Experimental scheme

Vehicle is water; Adjuvant is GLA-SE; Vaccine is ID83 + GLA-SE **Wks 12+5 and 12+10 indicate the time point following 12 weeks of treatment plus 5 and 10 weeks of follow-up without any treatment.

[0174] Pathological assessment. Photographs of representative lungs were taken after 72 hours of incubation in sterile PBS, and before homogenization for quantitative cultures, to record the macroscopic appearance of lung lesions. The other lung was washed and placed in 10% neutral-buffered formalin solution. These lungs were embedded in paraffin, sectioned, fixed on glass slides and stained with H&E and an acid-fast stain for review of

histopathology.

[0175] Histopathology Assessment. CFU counts (x) were log-transformed as (x+l) before analysis. Group means were compared by one-way analysis of variance with Bonferroni's post-test.

Results

[0176] Lung CFU counts during treatment. The mean (+ SD) lung log ₁₀ CFU count at D-26 was 2.78 + 0.21. The mean CFU count at the start of treatment (DO) was 7.60 + 0.23. After 6 weeks of treatment, lung CFU counts declined by approximately 0.9 logio in the

vehicle+adjuvant and vehicle+vaccine groups, whereas the CFU counts in the RHZ+saline, RHZ+adjuvant and RHZ+vaccine groups fell by 4.27, 4.19 and 4.44 log ₁₀ , respectively (Figure 6A). After 12 weeks of treatment, the CFU counts in the drug vehicle-treated groups were largely stable, whereas 3 of 5, 2 of 4, and 4 of 5 mice treated with antibiotics alone (RHZ+saline), antibiotic plus TLR4 adjuvant (RHZ+adjuvant), and antibiotics +ID 83 vaccine (RHZ +Vaccine), respectively, were culture-negative (Figure 6).

[0177] Lung CFU counts after treatment completion. After 5 weeks of followup post cessation of any treatment, 4 of 5 mice in each group had positive lung cultures (Table 3 below). The mean lung log 10 CFU counts in RHZ+saline, RHZ+adjuvant and RHZ+vaccine groups were 1.14 + 1.14, 0.72 + 0.49 and 0.63 + 0.42, respectively. Excluding the culture- negative mice, the CFU counts were 1.42 + 1.09, 0.90 + 0.32, and 0.78 + 0.27, respectively. After 10 weeks of follow-up, only 1 of 5 mice in the RHZ+vaccine group had a positive culture, compared to 3 of 5 mice in the RHZ+adjuvant and RHZ+saline groups. Excluding the culture-negative mice, the mean lung log 10 CFU counts in RHZ+saline, RHZ+adjuvant and RHZ+vaccine groups were 1.82 + 1.85, 1.37 + 1.56 and 0.75 + 1.68, respectively. At ten weeks after termination of treatment (Figure 7), the CFU counts in the three animals with positive lung cultures in both groups treated with RHZ+saline or RHZ+adjuvant alone exhibited logarithmic bacterial regrowth, while the only animal with positive lung culture in the RHZ+vaccine group exhibited bacterial regrowth that was reduced compared to the other two groups.

[0178] While the study was not powered for statistical significance in terms of CFU counts, these data demonstrated that treatment of an active tuberculosis according to the methods of the invention results in a durable improvement in a sign or symptom of tuberculosis and provide for a shortening of the duration of chemotherapy compared antibiotic drug therapy alone.

Table 3. Percentage (proportion) of mice with positive cultures

after 12 weeks of treatment followed by 5 or 10 weeks of follow-up

[0179] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

[0180] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Exemplar Amino Acid Sequences

ID93 fusion polypeptide with optional His tag (SEP ID NO: 1)

MGSSHHHHHHSSGLVPRGSHMTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVL

TASDFWGGAGSAACQGFITQLGRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGS

SWAGTHLANGSMSEVMMSEIAGLPIPPIIHYGAIAYAPSGASGKAWHQRTPARAEQV

ALEKCGDKTCKVVSRFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIVN

WACNELMTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSG

MAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSSVDIN

FAVLPPEVNSARIFAGAGLGPMLAAASAWDGLAEELHAAAGSFASVTTGLAGDAW

HGPASLAMTRAASPYVGWLNTAAGQAAQAAGQARLAASAFEATLAATVSPAMVA

ANRTRLASLVAANLLGQNAPAIAAAEAEYEQrWAQDVAAMFGYHSAASAVATQLA

PIQEGLQQQLQNVLAQLASGNLGSGNVGVGNIGNDNIGNANIGFGNRGDANIGIGNI GDRNLGIGNTGNWNIGIGITGNGQIGFGKPANPDVLVVGNGGPGVTALVMGGTDSL

LPLPNIPLLEYAARFITPVHPGYTATFLETPSQFFPFTGLNSLTYDVSVAQGVTNLH TA

IMAQLAAGNEVVVFGTSQSATIATFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPDG

GILTRFGFSIPQLGFTLSGATPADAYPTVDYAFQYDGVNDFPKYPLNVFATANAIAG I

LFLHSGLIALPPDLASGVVQPVSSPDVLTTYILLPSQDLPLLVPLRAIPLLGNPLAD LIQ

PDLRVLVELGYDRTAHQDVPSPFGLFPDVDWAEVAADLQQGAVQGVNDALSGLGL

PPPWQPALPRLFST

ID93 fusion polypeptide (SEP ID NO:2

MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQGFITQL

GRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWAGTHLANGSMSEVMMSEI

AGLPIPPIIHYGAIAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCG

AVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIVNWACNELMTSRFMTDPHAMR

DMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNI

VNMLHGVRDGLVRDANNYEQQEQASQQILSSVDINFAVLPPEVNSARIFAGAGLGP

MLAAASAWDGLAEELHAAAGSFASVTTGLAGDAWHGPASLAMTRAASPYVGWLN

TAAGQAAQAAGQARLAASAFEATLAATVSPAMVAANRTRLASLVAANLLGQNAPA

IAAAEAEYEQRVAQDVAAMFGYHSAASAVATQLAPIQEGLQQQLQNVLAQLASGNL

GSGNVGVGNIGNDNIGNANIGFGNRGDANIGIGNIGDRNLGIGNTGNWNIGIGITGN G

QIGFGKPANPDVLVVGNGGPGVTALVMGGTDSLLPLPNIPLLEYAARFITPVHPGYT

ATFLETPSQFFPFTGLNSLTYDVSVAQGVTNLHTAIMAQLAAGNEVVVFGTSQSATI A

TFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPDGGILTRFGFSIPQLGFTLSGATPA DA

YPTVDYAFQYDGVNDFPKYPLNVFATANAIAGILFLHSGLIALPPDLASGVVQPVSS P

DVLTTYILLPSQDLPLLVPLRAIPLLGNPLADLIQPDLRVLVELGYDRTAHQDVPSP FG

LFPDVDWAEVAADLQQGAVQGVNDALSGLGLPPPWQPALPRLFST

ID83 fusion polypeptide with optional His tag (SEP ID NO:3

MGSSHHHHHHSSGLVPRGSHMGTHLANGSMSEVMMSEIAGLPIPPIIHYGAIAYAPS

GASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCGAVAYNGSKYQGGTGLT

RRAAEDDAVNRLEGGRIVNWACNELMTSRFMTDPHAMRDMAGRFEVHAQTVEDE

ARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDAN

NYEQQEQASQQILSSVDINFAVLPPEVNSARIFAGAGLGPMLAAASAWDGLAEELHA

AAGSFASVTTGLAGDAWHGPASLAMTRAASPYVGWLNTAAGQAAQAAGQARLAA

SAFEATLAATVSPAMVAANRTRLASLVAANLLGQNAPAIAAAEAEYEQIWAQDVAA

MFGYHSAASAVATQLAPIQEGLQQQLQNVLAQLASGNLGSGNVGVGNIGNDNIGNA

NIGFGNRGDANIGIGNIGDRNLGIGNTGNWNIGIGITGNGQIGFGKPANPDVLVVGN G

GPGVTALVMGGTDSLLPLPNIPLLEYAARFITPVHPGYTATFLETPSQFFPFTGLNS LT

YDVSVAQGVTNLHTAIMAQLAAGNEVVVFGTSQSATIATFEMRYLQSLPAHLRPGL

DELSFTLTGNPNRPDGGILTRFGFSIPQLGFTLSGATPADAYPTVDYAFQYDGVNDF P

KYPLNVFATANAIAGILFLHSGLIALPPDLASGVVQPVSSPDVLTTYILLPSQDLPL LV

PLRAIPLLGNPLADLIQPDLRVLVELGYDRTAHQDVPSPFGLFPDVDWAEVAADLQQ

GAVQGVNDALSGLGLPPPWQPALPRLFST

ID83 fusion polypeptide (SEP ID NP:4

HLANGSMSEVMMSEIAGLPIPPIIHYGAIAYAPSGASGKAWHQRTPARAEQVALEKC GDKTCKVVSRFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIVNWACNE LMTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATS

LDTMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQQEQASQQILSSVDINFAVLPPE

VNSARIFAGAGLGPMLAAASAWDGLAEELHAAAGSFASVTTGLAGDAWHGPASLA

MTRAASPYVGWLNTAAGQAAQAAGQARLAASAFEATLAATVSPAMVAANRTRLA

SLVAANLLGQNAPAIAAAEAEYEQrWAQDVAAMFGYHSAASAVATQLAPIQEGLQQ

QLQNVLAQLASGNLGSGNVGVGNIGNDNIGNANIGFGNRGDANIGIGNIGDRNLGIG

NTGNWNIGIGITGNGQIGFGKPANPDVLVVGNGGPGVTALVMGGTDSLLPLPNIPLL

EYAARFITPVHPGYTATFLETPSQFFPFTGLNSLTYDVSVAQGVTNLHTAIMAQLAA G

NEVVVFGTSQSATIATFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPDGGILTRFGF SI

PQLGFTLSGATPADAYPTVDYAFQYDGVNDFPKYPLNVFATANAIAGILFLHSGLIA L

PPDLASGVVQPVSSPDVLTTYILLPSQDLPLLVPLRAIPLLGNPLADLIQPDLRVLV EL

GYDRTAHQDVPSPFGLFPDVDWAEVAADLQQGAVQGVNDALSGLGLPPPWQPALP

RLFST

Ryl813 (SEQ ID NO:5

MrfNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSEIAGLPIPPIIH Y

GAIAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCGAVAYNGSKY

QGGTGLTRRAAEDDAVNRLEGGRIVNWACN

Rv3620 (SEP ID NO:6

MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSL DTMTQMNQ AFRNIVNMLHG VRDGLVRD ANN YEQQEQ AS QQILS S

Rv2608 (SEP ID NO:7

MNFAVLPPEVNSARIFAGAGLGPMLAAASAWDGLAEELHAAAGSFASVTTGLAGD

AWHGPASLAMTRAASPYVGWLNTAAGQAAQAAGQARLAASAFEATLAATVSPAM

VAANRTRLASLVAANLLGQNAPAIAAAEAEYEQnVAQDVAAMFGYHSAASAVATQ

LAPIQEGLQQQLQNVLAQLASGNLGSGNVGVGNIGNDNIGNANIGFGNRGDANIGIG

NIGDRNLGIGNTGNWNIGIGITGNGQIGFGKPANPDVLVVGNGGPGVTALVMGGTDS

LLPLPNIPLLEYAARFITPVHPGYTATFLETPSQFFPFTGLNSLTYDVSVAQGVTNL HT

AIMAQLAAGNEVVVFGTSQSATIATFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPD

GGILTRFGFSIPQLGFTLSGATPADAYPTVDYAFQYDGVNDFPKYPLNVFATANAIA G

ILFLHSGLIALPPDLASGVVQPVSSPDVLTTYILLPSQDLPLLVPLRAIPLLGNPLA DLIQ

PDLRVLVELGYDRTAHQDVPSPFGLFPDVDWAEVAADLQQGAVQGVNDALSGLGL

PPPWQPALPRLF

Rv3619 (SEP ID NP:8

MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQGFITQL GRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWA