PREVENTION AND TR ^' EA TMENT OF MYCOBACTERIUM TUBERCULOSIS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/974,258, filed on April 2, 2014; the entire content of this application is incorporated herein in its entirety by this reference.

GOVERNMENT i NPING

This invention was made with government support under Grants ROI ΑΪ022553,

R01 .41047868, P50 AR063020 awarded by the National institutes of Health. The U.S. government has certain rights in the invention. This statement is included solely to comply with 37 C.F.R. § 401.14(a)ir)(4) and should not be taken as an assertion or admission that the application discloses and/or claims only one invention.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a global disease, with 8.7 million new eases and 1.4 million deaths in 20.11 (.1). In the US, estimates are that 10-15 million people are infected with Mycobacterium tuberculosis (Mtb) (2, 3), About one third of the world ^' s population are thought to harbor latent or persistent tuberculosis infection (1), which refers to those individuals who are infected with M, tuberculosis but do not have active disease. The recent emergence of multidrug resistant«( DR) and extensivel drug resistant- (XDR) TB in individuals in more than 70 countries is an emerging global threat (4). Persons infected with TB are often asymptomatic and can. be in a latent stage of the disease for a considerable period of time. .In its active state, the disease often manifests as an acute inflammation of the lungs, resulting in fever and a nonproductive cough. If untreated, serious complications and death typically result In 2012, 8.6 million people were newly infected with TB and 1.3 million people died from the disease, according to figures from the World Health

Organization (WHO). Major goals of WHO'S "Global Plan to Stop TB" include halting and beginning to the reverse the epidemic by 2015, as well as halving TB prevalence and death rates by 2015, compared, with 1990 levels [World Health Organization. 2013. WHO Global Tuberculosis Report 2013; World Health Organization. 2010. The Global Plan to Stop TB 201 1-2015 (Fact sheet}; Lonnroth K, et aL Lancet. 201 ; 375(9728): 181 -291 Currently available diagnostic technologies, including sputum-based smear microscopy and culture, are time- and labor- intensive, are often inaccurate, and are unable to distinguish between persons in the latent stage of the disease and those in the active stage. Moreover, smear has limited utility in atients with paueibaciliary TB and those unable to produce sputum samples (frequently pediatric and HIV-infected patients). In young children, testing thus often requires repeated gastric lavage, an invasive and

unpleasant method of sample collection. In addition to these limitations, approaches based on sputum and/or gastric aspirates do not diagnose extra-pulmonary disease. The lack of appropriate diagnostic tools remains a major obstacle for TB control in low-income countries. This has underscored the urgency in understanding the immunopathogenesis of human tuberculosis and development of new strategies for prevention and treatment.

Therefore,, there is an urgent need for new TB diagnostics and therapeutics that can be used in settings with resource constraints, and to identify biomarkers for effective treatment and diagnostic assays that detect active and latent infection by Mtb bacteria. SUMMARY OF THE INVENTION

The invention described herein provides methods for determining a M tuberculosis infection in patient using an interieukin-32 (I ^' L-32) biomarfcer. The invention further provides therapies lor preventing, mitigating, treating, or ameliorating a M. tuberculosa infection in a patient with an agent, such as a vaccine that boosts the levels of IL-32 alone or in combination with an agent that boosts levels of 25 hydroxyvitamin D (25D). .Provided herein are method for monitoring a latent or active state of tuberculosis infectio in a patient to thereby determine the progression of the Mycobacterium tubercuhsis infection.

One aspect of the invention relates to a method for determining whether a patient has a Mycobacterium tuberculosis infection, comprising: a) obtaining a biological sample from the patient; b) determining the level of interieukin-32 (IL-32) in the biological sample; and

c) comparing the level of IL-32 in the sample with the level of IL-32 in. a control sample, to thereby determine the state of the Mycobacterium tubercuhsis infection in the patient.

Alternati vely, the invention relates to method for determining whether a patient has a .Mycobacterium tuberculosis infection, comprising: a) determining the level of interieukin-32 (IL-32) in a biological sample of the patient; and b) comparing the level of IL-32 iii the sample with the level of IL-32 in a control sample, to thereby determine the state of the Mycobacterium tuberculosis infection in the patient.

Analogously, the invention relates to a method for determining whether a patient has a Mycobacterium tuberculosis infection, comprising: a) receiving a level of interieukin- 32 (IL-32) measured in a biological sample of the patient; and h) comparing the level of IL-

32 in the sample with the level of IL-32 in a control sample, to thereby determine the state of the Mycobacterium tuberculosis infection in the patient.

in certain embodiments, the control sample comprises a biological sample from a healthy patient within the same geographical, ethnic, or racial population as the patient, Alternatively, the level of IL-32 in control sample may be a level representative of one or more healthy patients within the same geographical, ethnic, or racial population as the patient (e.g., a mean or median level).

In certain embodiments, a higher level of IL-32 in the biological sample relative to the control sample indicates that the patient has a latent M. tuberculosis infection. In certain such embodiments, the method may further comprise terminating or reducing a medical treatment for an active tuberculosis infection, and/or commencing administration of a medical treatment for a latent tuberculosis infection.

A medical treatment for an active tuberculosis infection may comprise

administering an oral medicament for tuberculosis, such as administering one or more of, preferably a combination of all of. INH, RIF, pyrazmaraide (PZA), and ethambutol (EMB).

The oral medicament may be administered daily, twice weckiy, or thrice weckiy for at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least ten weeks, at least twelve weeks, at least fourteen weeks, at least sixteen weeks, or at least eighteen weeks. In certain such

embodiments, daily regimen of an oral medicament may comprise a dose of about 5 mg to about 300 mg of INH, about 10 mg to about 120 mg of RIF, about 1000 mg to about 2900 mg PZA, and about 800 mg to about 1600 mg of EMB. in certain such embodiments, a twice weekly regimen of an oral medicament may comprise a dose of about 5 mg to about

900 tug of ΪΝΗ, about 1 mg to about 600 nig of RIF, about 2000 mg to about 4000 mg PZA, and about 2000 mg to about 4000 mg of EMB. In certain such embodiments, a thrice weekly regimen of an oral medicament may comprise a dose of about 5 rag to about 900 mg of INH, about 1 mg to about 600 mg of RIF, about 1500 mg to about 3000 mg PZA, and about Ϊ 200 mg to about 2400 rag of EMS. A treatment for a latent tuberculosis infection may comprise administering an oral medicament for tuberculosis, e.g., isoniazid (INH), isoniazid (INH) and rifapentine (RPT), or rifampin (RIF), The ora! medicament may be administered daily, weekly, or two times a week for at least three months, at least four months, at least five months, at least si 5 months, at least seven months, at least eight months,, or at least nine months. In certain such embodiments, a daily regimen of an oral medicament may comprise a dose of about 5 mg to about 300 mg of INH for nine or six months or about 10 mg to about 120 nig of RIF for four months. In certain such embodiments, a weekly regimen of an oral medicament may comprise a dose of abou 50 mg to about 900 mg of!NM in combination with about it ⁾ 300 mg to about 900 mg of RPT, In certain such embodiments, a twice weekly regimen of an oral medicament may comprise a dose of about 15 mg to about 900 mg of INH for nine or six months. In certain such embodiments, the method may further comprise

administering a higher dose of the oral medicament for tuberculosis, e.g. , not to exceed a daily dose of up to about 300 mg of INH or about 600 mg of RIF, a twice weekly dose of

15 up to about 900mg of INH, or a weekly dose of up to about 900 mg of INH in combi nation with about. 900 rag of RPT. In certain embodiments, a lower level of IL-32 in the biological sample relati ve to the control sample indicates that the patient has an active Mycobacterium tuberculosis infection. In certain such embodiments, the method may further comprise terminating or reducing a medical treatment for a latent tuberculosis infection or

20 commencing or augmenting administration of a medical treatment for an acti ve tuberculosis infection, in certain such embodiments, a higher dose of a therapeutic agent to increase the level of IL-32 or boost the levels of hydroxy vitamin D ma be desirable. Alternatively, additional disease management therapies to improve symptoms of tuberculosis ma be administered to further increase the patient's levels of IL-32 or bi ©markers.

25 Another aspect of the invention provides a method for monitoring a latent state of

M. tuberculosis infection in a patient, comprising: a) obtaining a first biological sample from the patient; b) determining the level of IL-32 in the first biological sample; c) obtanung a second biological sample from the patient; d) determining the level of IL-32 in the second biological sample; and e.i comparing the level of IL-32 in the second biological 0 sample to the level of IL-32 in the first biological sample, to thereby determine the

progression of the Mycobacterium tuberculosis infection.

Alternatively, the invention pro vides a method for monitoring a latent state of M. tuberculosis infection in a patient, comprising: a) determining a level of IL-32 in a first bioiogical sample of the patient; b) determining a level of IL-32 in a second bioiogical sample of the patient; and e) comparing the level of IL-32 m the second biological sample to the level of IL-32 in the first biological sample, to thereby determine the progression of the Mycobacterium tuberculosis infection.

Analogously, the invention provides a method for monitoring a latent state of M. tuberculosis infection in a patient, comprising: a) receiving a level of iL-32 measured in a first biological sample of the patient; b) receiving a level of IL-32 measured in a second biological sample of the patient; and e) comparing the level of IL-32 in the second biological sample to the level of IL-32 in the first biological sample, to thereby determine the progression of the Mycobacterium tuberculosis infection.

In certain embodiments, a decrease in the levels of IL-32 in the second biological sample relative to the level of IL-32 in the first biological sample is indicati ve that the Mycobacterium tuberculosis infection is progressing to an acti ve state. In certain such embodiments, the method may further comprise commencing administration of or augmenting a medical treatment for an active tuberculosis infection or terminating or reducing a medical treatment for a latent tuberculosis infection.

in certain embodiments, a similar or increased level of IL-32 in the second biological sample relative to the level of IL-32 in. the first biological sample is indicative that the Mycobacterium tuberculosis infection remains in a latent state. In certain such embodiments, the method may further comprise terminating or reducing a medical treatment for an active tuberculosis infection, or commencing administration of a medical treatment for a latent tuberculosis infection.

Another aspect of the invention provides for a method for monitoring an active state of tuberculosis infection in a patient, comprising; a) obtaining a frrst biological sample from the patient; b determining the level of IL-32 in the first biological sample; c) obtaining a second biological sample from the patient; d) determining the level of IL-32 in the second biological sample; and e) comparing the level of IL-32 in the second biological sample to the level of IL-32 in the first biological sample, to thereby determine the progression of the Mycobacterium tuberculosis infection,

Alternatively, the invention provides for a method for monitoring an active state of

M. tuberculosis infection in a patient, comprising: a) detemuning a level of IL-32 in a first bioiogical sample of the patient; b) determining a level of IL-32 in a second biological sample of the patient; and c) comparing the level of IL-32 In the second bioiogical sample to the ievei of IL-32 in the first biological sample; to thereby determine the progression of the Mycobacterium tuberculosis infection.

Alternatively, the invention provides for a method for monitoring an acti ve state of M. tuberculosis infection in a patient, comprising: a) receiving a level of IL-32 measured in a first biological sample of the patient; b) receiving a level of IL-32 measured in a second biological sample of the patient; and c) comparing the level of IL-32 in the second biological sample to the level of IL-32 in the first biological sample, to thereby determine the progression of the Mycobacterium tuberculosis infection.

hi certain embodiments, the patient's IL-32 level is low if the patient's IL-32 level is lower than the level of IL-32 in a control population. The control population may be representative of healthy individuals within the same geographical, ethnic, or racial population as the patient.

in certain embodiments, the second sample was obtained at least one day, one week, or one month after the first sample was obtained, in certain embodiments, the second sample is obtained at least six months or even at least a year after the first sample was obtained.

in certain embodiments, a decrease in the levels of IL-32 in the second biological sample relative to the level of IL-32 in the first biological sample is indicative that the Mvcohacteritm tuberculosis infection is worsening. In certain such embodiments, the method may further comprise administering a medical treatment for tuberculosis or administering a stronger medical treatment for tuberculosis.

in certain embodiments, a similar level of IL-32 i the second biological sample relative to the level of IL-32 in the first biological sample is indicative that the

Mycobacterium tuberculosis infection remains in an active state. In certain such

embodiments, the method may further comprise commencing administration of or augmenting a medical treatment for tuberculosis.

in certain embodiments, an increased level of IL-32 in the second biological sample relati ve to the level, of IL-32 in the first biological sample is indicative that the

Mycobacterium tuberculosis infection is being successfully treated or is progressing to a latent state. In certain such embodiments, the method may further comprise terminating or reducing a -medical treatment for an active tuberculosis infection, or and/or commencing administration of a medical treatment for a latent tuberculosis infection.

In certain embodiments, determining comprises measuring an IL-32 mR A level. in certain embodiments, determining comprises measuring art IL-32 UNA level. In certain embodiments, determining comprises measuring an IL-32 protein expression level .

in certain embodiments, determining comprises using a .microarray.

In certain embodiments, the biological sample is a sample selected from blood, urine, spinal fluid, pleural fluid, saliva, or feces.

Another aspect of the invention relates to a method for preventing, mitigating, treating, or ameliorating M. tuberculosis infection in a patient, comprising: a) measuring an IL-32 level, e.g., in a biological sample of the patient, and, if the level is low, b) administering to the patient a pharmaceutically effective amount of a composition comprising an agent, such as a vaccine, that boosts the level of l ^' L-32.

Alternatively, the invention relates to a method for preventing, mitigating, treating, or ameliorating a M. tuberculosis infection in a patient, comprising: a) receiving an l ^' L-32 level measured in a biological sample of the patient, and, if the level is low, b)

administering to the patient a. pharmaceutically effective amount of a composition comprising an agent, such as a vaccine, that boosts the level of IL-32.

in certain embodiments, the patient has an active nikereulom infection.

in certain embodiments, the patient has a latent M. tuberculosis infection.

in certain embodiments, the method further comprises measuring a post-treatment IL-32 level, e.g., in a biological sample of the patient, and administering additional agent if necessary to further increase the patient's IL-32 level. Alternatively, the method may farther comprise receiving measured post-treatment IL-32 level, and administering additional agent if necessary to further increase the patient's IL-32 level. The post- treatment level may be measured at least a day, at least two days, at least three days, at least four days, at least five days, at least six days, at least a week, at least two weeks, at least three weeks, at least four weeks, or at feast a month after administering the agent that boosts the level of IL-32.

In certain embodiments, the method further comprises measuring the 25.D level, e.g., in a biological sample of the patient, and, if the level is low, e.g., less than about 30 iig/mL, administering an agent that boosts levels of 250 to the patient, e.g., to a level from about 40 ng/mL to about 100 ng/niL. Alternatively, the method may further comprise receiving a 25D level measured in the patient, and, if the level is low, administering an agent that boosts levels of 25D to the patient. The 25D level may be measured simultaneously with (e.g., from the same sample) as the IL-32 level, or may be measured at a different time and/or from a different sample. The patient ma also recei ve a medical treatment for an active tuberculosis infection.

In certain embodiments, the method further comprises measuring the post-treatment 25D level and administering additional agent that boosts levels of 25 D if necessary to further increase the patient's 25D level. Alternatively, the method may further comprise receiving a measured post-treatment 25D level, and administering additional agent that boosts levels of 25.D if necessary to further increase the patient's 25D level. The post- treatment level may be measured at least a day, at least two days, at least three days, at least four days, at least five days, at least six days, at least a week, at least two weeks, at least three weeks, at least four weeks, or at least a month after administering the agent that boosts the level of IL-32.

in certain embodiments, the agent is selected from an IL-32 protein, DMA, or RNA.

In certain embodiments, the infection is not resistant to antibiotics.

in certain embodiments, the agent induces an antibiotic response.

in certain embodiments, the antibiotic response is an increase in the concentration, expression level, or acti vity of one or more Vitamin D-dependent genes selected from Vitamin D receptor (VDRj, CYF27B 1 (or vitamin D i -a-hydroxy!ase), CD40, CYBB, or IL 5.

in certain embodiments, the antibiotic response is an increase in the concentration,, expression level, or activity of one or more genes selected from eathehcidin, DEFB4, IFIHI , APOBEC3G, C3, CFB, MI, CCL13, CCL22, PPBP, CCL8, APOL1, TAP2, CCL24, NDST1, TAP J , TAPBP, CXCL10, PLA2G7, or IDOI .

in certain embodiments, an increase in the concentration, expression level, or activity 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.«, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40-fold relative to control indicates that the agent- is effective.

it ts contemplated that all embodiments described herein, including those described under different aspects of the invention, can be combined with one another where not specifically prohibited. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 contains sine panels, A-I, depicting common latent tuberculosis blood signature identifies IL-32 and IL32 is part of an IL- 15 -induced gene set differentially expressed in latent TB. Pane! A shows top genes by cohort that are more highly expressed 5 by fold-change in whole blood from latent TB (n - 88) over active TB patients (« - 72) (upper left) or healthy endemic controls (» ^■ ■■■■ 25) {upper right). Common gene signature found in all patient cohorts of latent TB vs. active or healthy TB (below). Panel B depicts raw intensity values for IL-32 mRNA expression in each dataset by cohort, red line indicates mean. Panel C depicts raw data of separate cohort of Iatent and active TB patients it ⁾ and other diseases in which TB was a differential diagnosis. Panel D shows IL-32 mRNA expression data from active TB patients undergoing standard chemotherapy treatment. *P < 0,05 by student's t-test vs. time zero unless otherwise indicated. **P < 0.05 by one-way ANOVA ***P < 0.05 by Rruskal-WalHs one-way analysts of variance as indicated. Panel E shows IL-32 higher in latent TB patients vs. patients with sarcoidosis. Raw intensity

15 values of IL-32 from Germany ' 12 cohort. ***P < 0.05 by Kruskal-Wallis one-way analysis of variance. Panel F shows IL-32 higher in iatent TB patients vs. patients with sarcoidosis. Ra w intensity values of IL-32 from Maertzdorf et al 2 12. Red line indicates mean value, P-vahte by unpaired student ^' s t-test Panel G shows schema of workflow for analysis of TB expression data sets from whole blood. Panel H shows overlap of entire IL15black

20 module with common latent TB blood genes derived from fold change (FC) rank analysis of latent TB, active TB, or healthy control patients versus WGCNA of active TB patients undergoing chemotherapy treatment (TB Rx). Hypergeometrie distribution P value for enrichment of genes indicated for each overlap. Panel I shows fold change expression of the eight common latent TB genes (rows) across all TB cohorts (columns) indicated inside

25 each box; green indicates FC >1.2 in iatent TB over active or healthy control patients.

Genes are ranked in order of consensus across the seven comparisons as indicated on right.

Figure 2 contains eleven panels, A- , depicting identification of IL-32 correlated gene modules during treatment of active TB and during differentiation of M i macrophages. Weighted gene co-expression analysis (WGCNA.) of gene expression profiles from iatent 0 TB, active TB, and active TB patient blood undergoing chemotherapy treatment (Panels A,B) or adherent PBMC cytokine-polarized macrophage by IL-I5, 1L-U), and IL- into Ml , M2a, and M2c macrophages, respectively (Panels C,l>). Panels A and C depict hierarchical cluster tree depicting co-expression modules identified after WGCNA. Modules correspond to branches and are labelled by colors as indicated under the free. Panels B and D show heatraap depicting correlation of each module eigengene (ME) to treatment condition with corresponding P values. Number of probe sets per each module indicated at beginning of each row, red indicates positive correlation, green inverse correlation. Lower panel in Panel D depicts correlation of treatments to !L-32 expression or eigengene of ail genes annotated by gene ontology term 'defense response" . Panel E shows adherent FBMC were treated with IFN-γ or IL-15 for 24 h and CYP27B1 gene expression was measured by qPCR (mean fold change -± SEM, ti - 4). Panel F shows monocyte derived macrophages (MDSVls) were transfected with siRNA oligos specific w II S (siiLl S), or nonspecific fsiCtri) and then treated with lFN-γ for 24 h, IL-15 and IL-32 niR A assessed by qPCR (mean fold change ± SEM, u = 4). *P < 0.05 by student's t-test compared to media unless otherwise indicated. Panel G shows IL-32 expression after treatment with MVA85a vaccine (Matsumiya ct al. 2 13). Healthy -volunteers were vaccinated with candidate TB vaccine, MVA85a, and unstimulated PBMC isolated at indicated clays and lysed for gene expression analysis on illumina niicroarrays as described in original study. Raw intensity values of IL-32 shown. Panel H shows ! -y expression after treatment with MVA85a vaccine ( atsumiya et al 2 13), Healthy volunteers were vaccinated with candidate TB vaccine, fVfVA85a. RNA was isolated for microarray expression analysis from unstimulated peripheral blood mononuclear cells at indicated draepoints post-vaccination as described in original study. Raw intensity values of IF -y m ^' RNA shown, red line indicates mean value. Panel I shows schema depicting the workflow for analysis of macrophage expression profiles. WGCNA of gene expression profiles from adherent peripheral blood mononuclear ceil (PBMC) eytokine-polariized macrophages by IL-15 (200 ng/mi), IL-10 { 10 ng/ml), and IL-4 (1 ϋ/ml) into M l, M2a, and M2c macrophages, respectively. Panel J shows top eighteen genes expressed in the blood of latent TB vs. active TB and latent TB vs. healthy controls by fold-change analysis. Average fold-change of genes (rows) across TB cohorts (columns) indicated inside each box, green indicates FC > 1.2 in latent TB over active or healthy controls. Genes are ranked in order of consensus across the seven comparisons as indicated on right Genes are filtered to .have higher expression in latent TB vs. active TB ^" > 3 studies and latent TB vs. healthy controls > 1 , Panel shows average fold-changes depicted across TB cohorts of the 14 gene overlap of IA TENl ^' km and III 5 black from Figure 1 G, green indicates FC > 1.2 in latent TB over active or healthy controls. Figure 3 contains three panels, A-C, depicting IL- 15 defense response network links IL-32 to the vitamin D antimicrobial pathway. Panel A shows cell-type

deconvoiution of genes from IL-32 correlated black ^' module identified in Panel 2D. Each row represents the expression of each gene across a reference da tasei from separated cell types. Color depicts fold change expression in a given ceil type, relative to its expression among the 23 oilier cell types, statistical sigatficaa.ee indicated by arrowheads. Panel B shows visualization, of IL- 15 induced defense response connectivity network by topological overlap (XX715). Only those genes expressed in myeloid cells (FDR < 0.05) determined by (A) and connected to IL-32 are labeled. Panel C shows deconvoiution of tan WGCNA module derived from active TB patients undergoing treatment. Cell-type deconvoiution of genes from IL-32 connected tan module identified in Panel 2.8. Each row represents the expression of each gene across a reference dataset from: separated cell types. Color depicts fold change expression in a given cell type, relative to its expression among the 23 other cell types. Statistical significance indicated by arrowheads.

Figure 4 contains eight panels, A-H, depicting that IL-32 is necessary and sufficient for the induction of the IFN-γ dependent vitamin D pathway. Panel A depicts adherent. PBMCs were treated with IFN-γ (1.3 ng/ml) or IL-15 (200 ng/ml) for 24 hours, and IL-32 gene expression was measured by qPCR (mean fold change ± SEM, « = 4). Panel B shows monocyte derived macrophages (MDMs) were transfected with si A oligos specific for ILI5 (siIL.15) or nonspecific (siCtrl) and then treated with IFN-γ ( 1.3 ng/ml) for 24 hours, and IL-15 and IL-32 mR As were assessed by qPCR (mean fold change ± SEM, rt ~- 4). (Panels C and F) Adherent PBMCs were stimulated with IL-32 (50 ng/ml), IF -γ (1.3 ng/ml), or IL-15 (200 ng/ml) for 24 hours in 10% fetal calf serum (FCS), and CYP27B1 (Panel C) or VDR (Panel F) expression was assessed by qPCR (mean fold change SEM, « ~ 5 to 7). Panel I> CY.P27bl activity measured by treating adherent monocytes with IL- 32 (100 ng/ml), IFN-γ (1.3 ng/ml), or IL- 15 (200 ng/ml) in 10% FCS for 48 hours and for an additional 5 hours with j ¾]25D3. The amount of con version to j ^J I:I j 1 ,25D3 was measured by high-performance liquid chromatography (H ^' PLC). Panel E shows MDMs were transfected with stl ^' L-32 or siCtrl and treated with IFN-g ( L3 ng/ml) for 24 hours. IL- 32 and CY.P27B1. gene expression was determined by qPCR (mean fold change « SEM, n = 7). P value by Student 's t test. Panel G shows expression of IL-32 in simulated adherent PBMC microarray. Adherent PB C from, four healthy donors were stimulated by IL- 15, IL- ^' iO, lL-4, or media alone for 24h, R A was extracted and. global gene expression

- I I - measured by Asymetrix Human IJ I 33 Piu 2.0 expression array- Expression of IL-32 shown as arbitrary units at 24b after simulation, red line indicates mean intensity. Panel If shows that IL-32 induction of CYP27bl was dose-dependent. Adherent PBMC were stimulated with indicated concentration of IL-32 for 24h in 10% PCS and CYP27BI

5 m A expression assessed by qPCR (mean fold change _· ÷■ SEM, n - 4 to 1.0).

Figure 5 contains four panels, A-D, depicting that IL-32 triggers a vitamin D- dependeut antimicrobial response against M. tuberculosis. Paael A shows human monocytes purified by negative selection were cultured in 10% vitamin .D-sufficient human scrum and stimulated with !L-32, IFN-γ, or IL- 15 for 24 h. Antimicrobiai peptides it ⁾ cathelicidin (Cath.) and DEFB4 mRNA expression was determined by qPCR (mean fold change ± SEM, » = 3 to 5). Panel B shows purified monocytes were pretreated with the VD antagonist VAZ (ZK 159222) for 15 min, and treated with IL-32 for 24 h. RNA expression of indicated genes measured by qPCR (mean fold change ± SEM, «™ 3). Fane! C shows human monocyte derived macrophages were infected with M. tuberculosis H37Rv

15 overnight. After infection, cells were treated with IL-32 or IFN-y for four days. Viability of M. tuberculosis was calculated by the ratio of bacterial 16S RNA and D A (IS 1 10) as measured by qPCR, and percent increase or decrease -relative to no treatment (media) was determined (mean fold change ± SEM, « *= 3). *P < 0.05 by student's t-test versus media unless otherwise indicated. Panel J) shows IFN-y induced DDX60 expression i unchanged

20 by silLiS knockdown. Monocy te-deri ved macrophages were transfeeted with siRNA oligos specific for lLlS (silLi S), or nonspecific (siCtri) and then treated with IFN-γ for 24 hours, DDX60 mRNA assessed by qPCR (mean fold change ± SEM, n - 4). P- alue by student's t-test.

Figure 6 depicts IFN-y induced TLR7 expression is unchanged by si!L32

25 knockdown. Monocyte-derived macrophages were transfeeted with siRNA ol igos specific for IL-32 (sil.L32), or nonspecific (siCtrl) and then treated with IFN-g for 24 hours, TLR7 mRNA assessed by qPCR (mean fold change ± SEM, n - 4), P-value by student's t-test.

Figure 7 depicts the role of IL-32 in host defense. This model shows the IL- i 5 dependent induction of IL-32 by IFN-y leads to dual pathways in which l ^' L-32 can enhance 0 antimicrobial activity of macrophages infected with M. tuberculosis. The current study demonstrates IL-32 can directly induce the vitamin D-dependent antimicrobial pathway, while previous studies have shown IL-32 triggers differentiation of dendritic cells that can cross -present to activate CD8+ T cells which are aiso abie to activate an antimicrobial response against M. tuberculosis.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides methods for determining an active or latent

M. tuberculosis infection in a patient using an IL-32 biomarker. The invention further provides therapies for preventing, mitigating, treating, or ameliorating a M. tuberculosis infection in a patient with an agent, such as a vaccine, that boosts the levels of 1L-32, alone or in combination with an agent that boosts levels of 25D (such as eholecaleiferol, calcifedioi, 25-hydroxycholecalciferol., or calcitriot that contributes to a patient's levels of 25D ). Provided herein are also methods for monitoring a latent or active state of M.

tuberculosis infection in a patient using IL-32 levels, to thereby determine the progression of the . tuberculosis infection. By comparing the relati ve increases or decreases of IL-32 levels of a first sample relati ve to a second sample obtained from the patient, may be used to monitor whether a M. fubercuh.m infection is progressing to an active state, remains in a latent state, worsening, progressing to a latent state, or being successfully treated.

Various aspects of the invention are described in further detail herein.

I. Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly - -understood by- those of ordinary skill in the art. Generally, nomenclature and techniques relating to chemistry, molecular biology, ceil and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

Throughout this specification, the word "comprise" or variations such as

"comprises" or "comprising" may be understood to impl the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components). The singular forms "a," "an," and "the" include the piorais itniess the context clearl dictates otherwise. The term ""including ^"' is used to mean ""including but not limited to," "including" and ""including but not limited to' ^" are used interchangeably.

"About" and "approximately"' shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical ly, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about"' and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately"' can be inferred when not expressly stated,

^' T e term "analogous" or "liomologoiss" amino acid sequences refer to amino acid sequences with sufficient identity to the human IL-32 sequences so as to possess the biological activity of eliciting a protective immune response to tuberculosis. For example, a« analogous or homologous peptide can be produced with "silent" changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues of human IL-32, yet tie peptide still possesses the function or biological activity of the full-length IL-32. Examples of such differences include additions, deletions or substitutions of residues of the amino acid sequence of human IL-32. Such peptides can be made by mutating (e.g., substituting, deleting or adding) one or more amino acid or nucleic acid residues to isolated IL-32 polypeptides. Said mutations can be performed using methods described herein and those known in the art. In particular, the present invention relates to homologous peptide molecules havin at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID Os: 1. 2, or 3. Homologous !L-2 peptides can be determined using methods known to those of skill in the art. Homology searches can be performed at NCBI against the GenSairk, EMBL and SwissProt databases using, for example, the BLAST network service. The term "percent identity" refers to the percentage of identical amino acids between two amino acid sequences. The term "percent similarity" refers to the percentage of similar or conservative amino acids between two amino acid sequences.

As used herein, "biological sample" means a sample of biological tissue or fluid that contains nucleic acids or polypeptides. Such samples are typically from humans, but tnelude tissues isolated from non-human primates, or rodents, e.g., mice, and rats.

Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, cerebral spinal fluid, blood, saliva, cerebral spinal fluid, pleural fluid, urine, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc., that can be used to measure expression level of a IL-32 gene, D A, A, or polypeptide. The term "combination" combination therapy," as used herein, refer to a first agent in combination with a second agent and includes co-administration of a first agent and a second agent, which for example may be dissol ved or intermixed in the same

pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent. The present invention, therefore, includes methods of combination therapeutic treatment and

combination pharmaceutical compositions,

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 die art of peptide chemistry would expect the secondary structure and hydropathic nature of the peptide to be substantially unchanged, in general, the following groups of amino acids represent conservative changes; (! ) Ala, Pro, Giy, Gin, Asp, Gin, Asn, Ser, Thr: (2) Cys, Ser, Tyr, Thr; (3) Val, lie, Leu, Met, Ala, Fhc; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His.

The term "dose," as used herein, refers to an amount of a therapeutic agent, such as an agent that boosts levels of 25D or an agent, such as a vaccine, to boost IL-32 levels, which is administered to a subject.

The term "dosing," as used herein, refers to the administration of a therapeutic agent, such as a vaccine, to boost I.L-32 levels, to achieve a therapeutic objective fe.g., treatment, prevention, mitigation, or amelioration of a tuberculosis infection). The level of dosing could be based on the baseline level of 1L-32. One way of determining an appropriate dose would be to measure baseline iL-32 or 25D levels in a control population to determine target dose, followed by additional measurements after administration to determine the dose's effect on IL-32 or 25D levels. For example, 25D levels or

concentrations of about 14ng/mL ± I ng/mL is insufficient or considered low or

therapeutically ineffective level, whereas 25 levels or concentrations of about 20 ng/mL ± 1 ng/fflL is sufficient or considered to be a therapeutically effective level.

A "dosing regimen" describes a schedule for administering a therapeutic agent, such as a. vaccine, to boost IL-32 levels, e.g., a treatment schedule over a prolonged period of time or throughout the course of treatment, e.g., administering a first dose of an agent to boost IL-32 levels at week 0 followed by a second dose of an agent to boost 11,-32 levels on. a weekly or biweekly dosing regimen.

As used herein, a "fragment" may refer to a biologically active portion of full-length human IL-32 protein. Fragments of full-length IL-32 protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence set forth in SEQ ID NOs: 2 or 3, which include fewer amino acids than the full length 1L-32 protein, and exhibit at least one activity of IL-32. Fragments of Mi-length l.L-32 protein retain their ability to elicit an immune response and may be adapted for immunotherapy. A fragment or biologically active portion of an IL-32 protein may be substantially identical to SEQ ID NOs; 2 or 3, and retains the functional activity of the protein of SEQ ID NOs: 2 or 3, yet differs in amino acid sequence clue to natural allelic variation or mutagenesis, in other embodiments, the !L-32 protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NOs: 2 or 3. In other embodiments, the IL-32 protein comprise an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99,4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NOs: 2 or 3.Those "in need of treatment" include mammals, such as humans, already having a latent or active tuberculosis infection, including those in which the disease or disorder is to be prevented, e.g., those identified as being at risk of developing the disease or disorder.

The term "'preventing" is art-recognized, and when used in relation to a medical condition such as a M. tuberculosis infection, is well understood in the art, and includes administration of a composition which reduces the frequency of. or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

The term "prophylactic" or "therapeutic" treatment is art-recognized and refers to administration of a drug to a host. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment ts therapeutic {i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom), Prophyiatie and therapeutic treatment may be used in conjunction with known methods of relieving M. tuberculosis infection .

The terms "subject" and "patient", as used herein, are used interchangeably. In certain embodiments, a subject refers to an indi vidual who ma be treated therapeutically with an agent to boost IL-32 levels. A "therapeutically effective amount" of an agent, such as a vaccine, to boost IL-32 levels, with respect to the subject method of treatment, refers to art amount of the compounds) in a preparation which- when administered as part of a desired dosage regimen to a subject achieves a therapeutic objective (e.g., treatment of a M. tuberculosis infection). A therapeutically effective amount may be determined by measuring baseline IL-32 or 25D levels to determine a target dose, followed by additional measurements after administration to determine the effect of the dose on IL-32 or 25D levels. In such embodiments, if the patient's IL-32 concentration, level, or activity is increased, then the dose is a therapeutically effective amount.

The term "variant" refers to a peptide that differs from the recited peptide only in conservative substitutions and/or modifications, such that the therapeutic, antigenic and or immunogenic properties of the peptide are retained, A variant of an IL-32 will therefore stimulates a host defense and/or antimicrobial pathway. Peptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% homology to the identified peptides. For peptides with im unoreaetive properties, variants can, alternatively, be identified by modifying the amino acid sequence of one of the abo ve peptides, and evaluating the ininMnoreactiviiy of the modiiicd peptide. Variants can also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the peptide. For example, a peptide can be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translaoonaily or ost- translationally directs transfer of the protein. Other N-terminal sequences may comprise the sequence set forth in SEQ ID NO: 1. The peptide can also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the peptide (e.g., poiy- His), or to enhance binding of the peptide to a solid support.

II. Agents to boost IL-32 levels

The present invention relates in part to therapeutics that boost a patient's levels of IL-32, such as vaccines comprising synthetic IL-32 peptides, proteins, fragments, variants, or homologues thereof that are generated by synthetic or recombinant means or can be purchased commercially, such as the BCG vaccine (14) of recombinant IL-32y from. R&D systems (Catalog no. 4690-IL/CF). Said recombinant IL-32y may comprise an -terminits having an amino acid sequence set forth in SEQ ID NO: 1 (MNHKVHHHHHH) fused to a C-terminus having an amino acid sequence comprising human iL-32 γ set forth in SEQ ID NO: 2 (MCFPKVLSDDMK LKAmVMLLPTSA<3K3LGAWVSACDTEDTVG

HLGPWRDKDPALWCQLCLSSQHQAIERFYDKMQ AESGRGQVMSSLAELEDDFK

EGYLETYAAYYEEQHFELTPi ,E ERDGLRCRGNRSPVF

VMRWQAMLQRLQTWWI-IGVLAWVKEKVVALVHAVQALWKQFQSFCCSLSELF

MSSFQSYGAPRGD EELTPQ CSEPQSSK) to generate a fusion iL-32y polypeptide having the amino acid sequence set forth in SEQ ID NO: 3

( NHKVHHHHHHM

VGeEGPWRDKDPALWCQLCLSSQHQAIERFYDK QNAESGRGQVMSSLAEL EDDF EGYLETVAAYYEEQHPELTPLLEKERDGLRCRGNRSPVPDVEDPATEEPGE SFCD VMRWFQAMLQRLQTWWHGVLAWV E VVALVHA.VQALWKQFQSFCCS

LSELFMSSFQSYGAPRGDKEELTPQ CSEPQSS ). in certain embodiments, the synthetic IL-32 vaccine comprises an IL-32 protein having an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%. 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9 or more identical to SEQ ID Os: 2 or 3. When administered, the agents boost IL- 32 le vels. Variants or homologues of a nati ve iL-32 can generally be prepared using standard mutagenesis techniques, such as oligonucieotide-directed site-specific

mutagenesis. Sections of the DNA sequence can also be removed using standard techniques to permit preparation of truncated peptides.

Agents such as recombinant IL-32 peptides, proteins, peptides containing portions and/or variants of a native IL-32 can be readily prepared from a D A sequence encoding the peptide using a variety of techniques well known to those of ordinary skill in the art. For example, supeniatants from suitable host/vector systems which secrete recombinant protein into culture media can be first concentrated using a commercially available filter. Following concentration, the concentrate can be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant protein.

Any of a variety of expression vectors known to those of ordinary skill in the art can be employed to express recombinant IL-32 or variants or homologues thereof. Expression can be achieved in any appropriate host cell that has been transformed or transfceted with an expression vector containing a DNA molecule that encodes a recombinant peptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic ceils. Host cells employed may be E. coli, yeast or a mammalian cell line such as COS or€110. The DNA sequences expressed in this manner can encode naturally occurring IL-32, portions of naturally occurring IL-32, or other variants thereof.

Uses of plasmids, vectors or viruses containing the cloned l.L-32 can include one or more of the following; ( I ) generation of hybridization probes for detection and measuring level of IL-32 in tissue or isolation of IL-32 homologs; (2) generation of IL-32 niR A or protein in vitro or in vivo; (3) generation of transgenic non-human animals or recombinant host ce to boost IL-32 levels.

The term ^pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount of the composition or therapeutic agent, such as a vaccine, to boost IL- 32 levels (or "IL-32 vaccine"), effective t M. tuberculosis infection in a patient, and/or effecting a beneficial and/or desirable alteration in the general health of a patient suffering from M tuberculosis infection. A "pharmaceutically effective amount" or "therapeutically effective amount" also refers to an amount that improves the clinical symptoms of a patient.

The phrase "pharmaceutically acceptable excipient" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, lubricant, binder, carrier, huraectant, disintegrant, solvent or encapsulating material, that one ski! led in the art would consider suitable for rendering a pharmaceutical formulation suitable for adrairri station to a subject, Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, as well as

"pharmaceutically acceptable" as defined above. Examples of materials which can serve as pharmaceutically acceptable excipients include but are not limited to: sugars, such a lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and Its derivatives, such as sodium earboxymetliyS cellulose, ethyl cellulose and cellulose acetate; powdered tragaeanth; malt; gelatin; talc; silica, waxes; oils, such as com oil and sesame oil; glycols, such as propylene glycol and glycerin; polyoSs, such as sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar;

buffering agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; and other non-toxic compatible substances routinely employed in pharmaceutical formulations.

The pharmaceutical formulation may comprise suitable excipients including pharmaceutically acceptable buffers, stabilizers, local anesthetics, and the like that are well known in the art. For parenteral administration, an exemplary formulation may be a sterile solution or suspension. For oral dosage, a syrup, tablet or palatable solution; for topical application, a lotion, cream:, spray or ointment; for mtravaginal or intrarectal administration, pessaries, suppositories, creams or foams. Preferably, the route of administration is parenteral, more preferably intravenous.

I alternative embodiments, a pharmaceutical composition of the invention may be in a form adapted for oral dosage, such as for example a syrup or palatable solution; a form adapted for topical application, such as for example a cream or ointment; or a form adapted for administration b inhalation, such as for example a microerystailine powder or a solution suitable for nebulization. ethods and means for formulating pharmaceutical ingredients for alternative routes of administration are well-known in the art, and it is to be expected that those skilled in the relevant arts can adapt these known methods to the IL-32 vaccine.

A tablet may be made by compression or molding, optionally with one or more accessor) ¹ ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropy!methyl cellulose), lubricant, inert diluent, preservative,

disintegrant (for example, sodium starch glycol ate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mix ture of the powdered compound moistened wi th an inert liquid diluent.

The tablets and other solid dosage forms of the pharmaceutical compositions of the present in vention may optionally be scored or prepared with coatings and shells, such a enteric coatings and other coatings well kno wn in the pharmaceutical -formulating art. They may also be formulated so as to provide slow or controlled release of the modified therein using, for example, hydroxypropyimethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by

incorporating sterilizing agents in the form of sterile solid compositions that can be dissol ved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The IL-32 vaccine cart also be in micro-encapsulated form, if appropriate, with one or more of the above-described exeipients.

Liquid dosage forms for oral administration of the IL-32 vaccine include pharmaceutically acceptable emulsions, raicroemuJsions, solutions, suspensions, syrups and elixirs. In addition to the IL-32 vaccines, the l quid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, so!ubilizmg agents and emulsifiers.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyi alcohols, polyoxyethykne sorbitol and sorbitan esters, microcrystaHine cellulose, aluminum metahydroxide, bentoatte, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise a IL-32 vaccine in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which, may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of

microorganisms may be ensured b the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanoi, phenol sorbic acid, and the like, it may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.

Examples of pharmaceutically acceptable antioxidants include but are not limited to ascorbic acid, cysteine hydrochloride, sodium metabisuSfite, sodium sulfite, ascorbyl palmitate, butyiated hydroxyanisole (BHA), butyiated hydroxy toluene (BHT), propyl gailate, alpha-tocopherol, and chelating agents such as citric acid, ethyienechamine tcfraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. injectable depot forms are made by forming microencapsule matrices of the subject compounds irt biodegradable polymers such as poiyiacti de-poly glycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employ cd, the rate of drug release can be controlled. Examples of other biodegradable polymers include

polyiorthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulstons that are compatible with body tissue.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The IL-32 vaccine may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or prope!lants thai may be required.

A pH-adjusting agent may be beneficial to adjust the pH of the compositions by including a pli-adjusting agent in the compositions of the in vention. Modifying the pli of a formulation or composition may have beneficial effects on, for example, the stability or solubility of a therapeutically effective substance, or may be useful in making a formulation or composition suitable for parenteral administration. pH-adjusting agents are well known in the art. Accordingly, the pH-adj listing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary pE-adjusiing agents that ma be used in the compositions of the invention. pH -adjusting agents may include, for example, acids and bases. In some embodiments, a pH-adjusting agent includes, but is not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof. The pH of the compositions of the invention may be any pH that provides desirable properties for the formulation or composition. Desirable properties may include, for example, therapeutically effective substance stability, increased therapeutically effective substance retention as compared to compositions at other pHs, and improved filtration efficiency. In some embodiments, the pH of the compositions of the invention may be from about 3.0 to about 9.0, e.g., from about 5.0 to about 7.0, in particular embodiments, the pH of the composition of the invention may be 5.5.40. l _s 5.6.40.1, 5.74.0.1, 5,8*0.. l _f 5.940.1 , 6.04.0.1, 6.140.1 , 6.2*0.1 , 6.3.40.1, 6.44.0,1, or 6.5*0.1 . in certain embodiments, the I.L-32 vaccine is prepared substantially efhanoi-free and suitable for parenteral administration. By substantially free of ethanol, it is meant that the compositions of the invention contain less than 5% ethanol by weight. In preferred embodiments the compositions contain less than 2%, and more preferably less than 0.5% eihanoi by weight. In certain embodiments, the compositions further comprise one or more pharmaceutically acceptable excipienis. Such compositions include aqueous solutions of the i.L-32 vaccine. In certain embodiments of such aqueous solutions, the IL-32 vaccine occurs a t a concentration of at least 7 rag mL, at least 10, or 15 or more mg/ml Any of such compositions are also substantially free of organic solvents other than ethanol.

A buffer may be used to resuspend the compound in solution. In certain

embodiments, a buffer may have a p a of, for example, about 5.5, about 6.0, or about 6.5. One of skill in the art would appreciate that an appropriate buffer may be chosen for inclusion in compositions of the in vention based on its pKa and other properties. Buffers are well known in the art. Accordingly, the buffers described herein are not intended to constitute an exhaustive list, but are provided merely as exemplars' buffers that may be used in the compositions of the invention. In certain embodiments, a buffer may include one or more of the following: Tris, Tris HQ, potassium phosphate, sodium phosphate, sodium citrate, sodium aseorbare, combinations of sodium and potassium phosphate, Tris Tris HO, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacod late, succinate, 2-(N-morpholmo)ethanesulfonic acid (MBS), raaleate, bis-tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof.

A soiubilizing agent may be added to increase the solubility of a drug or compound. In some embodiments, it may be beneficial to include a solubilizing agent to the IL-32 vaccine. Solubilmng agents may be useful for increasing the solubil ity of any of the components of the formulation or composition. The solubilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary solubiiizing agents that may be used in the compositions of the invention, in certain embodiments, soSubilizing agents include, but are not limited to, ethyl alcohol, tert-butyi alcohol, polyethylene glycol, glycerol, raethylparaben, propylparaben, polyethylene glycol, polyvinyl pyrrolidine, and any pharmaceutically acceptable salts and/or combinations thereof.

A stabilizing agent may help to increase the stability of a therapeutically effective substance in compositions of the invention. This may occur by, for example, reducing degradation or preventing aggregation of a therapeutically effective substance. Without wishing to be bound by theory, mechanisms for enhancing stability may include sequestration of the therapeutically effective substance from a solvent or inhibiting free radical oxidation of the anthracycUne compound. Stabilizing agents are well known in the art. Accordingly, the stabilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary stabilizing agents that may be used in die compositions of the invention. Stabilizing agents may include, but are not limited to, ernulsifiers and surfactants.

A surfactant may be added to reduce the surface tension of a liquid composition. This may provide beneficial properties such as improved ease of filtration. Surfactants also may act as emulsifying agents and/or solubiiizing agents. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary surfactants that may be used in the compositions of the invention. Surfactants that may be included include, but are not limited to, sorbitan esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poioxamers (i.e., pluronies), ethylene oxides and polyethylene oxides (eg., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

A tomcity-adjustmg reagent may be used to help make a .formulation or composition suitable for administration. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration. Toniciiy-adjusting agents are well known in the art. Accordingly; the foniciry-adjusting agents described heretri are not intended to constitute an exhaustive list, but are provided merely as exemplary tonicity-adjusting agents that may be used in the compositions of the invention. Tonicity-adjusting agents may be ionic or non-ionic and include, but arc not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

IIL Diagnostic and prognostic methods usin 1L~32 and hiomarkcrs of

interconnected pathways

The methods of the present invention are used in conjunction with measuring IL- leveis, concentrations, or activities alone or with biomarkers of 1L- 15 defense response network, vitamin D antimicrobial pathway, or IFN-γ dependent vitamin D pathway to measure the effect of treatment with an I.L-32 vaccine, alone or in combination with an agent that boosts levels of 25D. The biomarkers of IL- 15- induced host defense pathway in macrophages as determined by WGCNA include, but not limited to, genes involved in the vitamin D antimicrobial pathway (C YP27B 1„ CD40, CYBB„ IL 15). Additional set of genes implicated n antimicrobial responses was identiiled (IFl ^' Ml, APOBEC3G, C3, CFB, M1, IDOI). Other biomarkers include genes having MHC class 1 antigen presentation {TAP !., TAP2, TAPBP) and genes involved in lipid, metabolism (PLA2G7, APOL ^' i ). Assays for biomarker levels, concentrations, and activities may be performed according to the methods described herein and known in the art

in certain embodiments, a control sample from a healthy patient within the same geographical, ethnic, or racial population as the patient is used in determining whether a patient has a M tuberculosis infection, in. certain embodiments, a higher level of IL-32 in the biological sample of the patient relative to the control sample indicates that the patient. has a latent M. t berc bsis infection. In other embodiments, a lower level of i.L-32 in the biological sample relative to the control sample indicates that the patient has an active M. tuberculosis infection. A high level of IL-32 can be determined as a 0. 1 , 0.2, 0,3, 0,4, 0,5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1, 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 3, 4, or 5-foi.d increase in IL-32. RNA DNA, or protein levels relative to the IL-32. levels in a control sample, Likewise, a low level of IL-32 can be determined as a 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 3, 4, or 5-fold decrease m IL-32 RN A, DNA, or protein levels relative to the IL-32 levels in a control sample.

in the methods for monitoring a latent state in M. tuberculosis infection in a patient, a decrease of 0. i, 0.2, 0.3, 0,4, 0,5, 0.6, 0.7, 0.8, 0,9, 1 ,0, 1 , 1, 1 ,2, 1 ,3, 1 ,4, 1 ,5, 1 ,6, 1 ,7, 1 .8, 1 .9, 2, 3, 4, or 5-fold in the levels of IL-32 in the second biological sample relative to the level of IL-32 in the first biological sample is indicative that the Mycobacterium

tuberculosis infection is progressing to an active state, in the methods for monitoring a latent state in M. tuberculosis infection in a patient, a similar or increase of 0.1 , 0,2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1.9, 2, 3, 4, or 5-fold in the levels of iL-32 in the second biological sample relative to the level of I.L-32 in the first biological sample is indicative that the Mycobacterium tuberculosis infection remains in a latent state. in the methods For monitoring an active state in M. tuberculosis infection in a patient a decrease of 0.1. 0.2, 0.3, 0.4. 0.5. 0.6. 0.7. 0.8. 0.9. i .O. i .l . i .2. i .3. i .4. i .5. i .6. 1.7, L8, 1.9, 2, 3, 4, or 5-fold in the levels of IL-32 in the second biological sample relative to the level of IL-32 in the first biological sample is indicative that the Mycobacterium tuberculosis infection is worsening. In the methods for monitoring art active state in M. tuberculosis infection in a patient, an increase of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 3, 4, or 5-fold in the levels of IL-32 in the second biological sample relative to the level of IL-32 i the first biological sample is indicative that the Mycobacterium tub ^' erctihsis infection is being successfully treated or is progressing to a latent state, in the methods for monitoring an active state in M

tuberculosis infection in a patient, a similar level of IL-32 in the second biological sample relative to the level of IL-32 in the first biological sample is indicative that the

Mycobacterium tuberculosis infection remains in an active state.

Determining the level of IL-32 may be combined with the detection of one or more other biomarkers including, but not limited to, CYP27B 1, CD40, CYB.8, IL15, 1 ^' FIHl , APOBEC3G, C3, CFB, NMl, IDO L PLA2G7, APOLl, TAP!, TAP2, TAPBP, NDST1 , PPBP, CCLS, CCL13, CCL22, CCL24, CXCL!O, GP 56, GZMA, S1P 5, PPP2R2B, NKG7, PRF i , GPR56, RUNX3, CD247, GZMH, TGFBR3, FGFBP2, CD247, S.4MD3, PRKCH. SAMD3, SBKl, KLRDl , ΡΥΗΪΝΊ , HS.276860, IL32, SLAMF6, HOMES, RFT .1 _f IL2R.B, HCST, ONLY, KLRD I , PLEKHFl, PYHIN1 , PTPN4, MATK, STAT4, ADRB2, ARHGEF3, CCL4L2, G ^' NLY, STK39, HOPX, MAP4 1 , ZAP70, S APL SH2D 1 A, YWHAQ, CD8A, PRKCQ. CX3CRL LOC729495, IL10RA, GZMB, CX3C .L CDC25B, RALGDS, RAP2A, LOC653438, TARP, FLJ20699, KLRG 1 , KLRFl, CD8A, FAM108A2, O LY, SYTL2, COLN2, TTC38, LRF1 , GNPTAB, CCL5, SYTl 1, HS.19339, CTSW, CBLB, APOBEC3G, ST6GAL1, AUTS2, F1J 14213, LOC643035, KIR2DL3, K.LRC3, KIR2DL4, GPSL ZNF683, PATL2, MYOM2, D 5B, BCL-2, BCL9L, GATA3, LRP2, ZMIZ2, FBX07, VAMP2, EBF.I , PAOX, ΠΜ2Α, for which modulation i expression eorrelates with acti ve or latent M. tuberculosis infection or efficacy of treatment using IL-32 vaccines, (see Example 4 and Figure 3), One may select one or more of the aforementioned biomarkers for measurement in combination with IL-32. Similarly, three or tno.ee, four or more or five or more or a multitude of biomarkers can be used together for determining a treatment, diagnosis, or prognosis of a patient. Diagnostic kits may be provided for the detection of IL-32 peptides or the quantification of these peptides,, having antibodies specific for them or a portion thereof. Kits may further contain detection reagents, such as antibodies, to any one of the aforementioned biomarkers. Any combination of detection reagent s specific for the IL-32 peptide and biomarker may be used in multiplex diagnostic kits. Kits may further comprise positive or negative 11,-32 peptide and biomarker standards, wherein the standards are obtained from control or healthy patients, patients having latent tuber f sis infection, or patients having acti ve M iiibercuki infection ,

In the methods for preventing, mitigating, treating, or ameliorating a tuber iosis infection in a patient: administered with a pharmaceutically effective amount of a

composition comprising an agent, such as a vaccine, that boosts the level of IL-32, the post- treatment levels of one or more biomarkers may increase relative to the pre -treatment levels. In certain embodiments, the levels of at least one biomarker CYP27B1, CD40 CYBB, IL15, IFIHI , APOBEC3G, C3, CFB, NML IDOl, PLA2G7, APOLL TAP I., TAP2, TAPBP, NDSTI , PPBP, CCL8, CCL13, CCL22, CCL24, or CXCL10 will increase by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, , 7, 8, , 10, 15, 20, 25, 30, 35, 0- fold in patients gi ven a dose of IL-32 vaccine relati ve to the biomarker level in an untreated patient or a patient treated with placebo,

IV. IL-32 and biomarker protein level detection techniques

Methods for the detection of protein, e.g.. IL-32 protein and biomarkers, such as

CYP27B5 , CD40, CYBB, IL55, IFIHI , APOBEC3G, C3, CFB, NML IDOL PLA2G7, APOLL TA 1 , TA.P2. TAPBP, NDSTI , PPBP, CCL8, CCL13. CCL22, CCL24, CXCLI O, GPR56, GZMA, S IPR5, PPP2R2B, K.G7, P FL GP 56, RUNX3, CD247, GZM.H, TGFBR3, FGFBP2, CD247, SAMD3, PRKCH, SAMD3, SB 1 , LRD1, PYRIN L HS .276860, 1132, SLAMF6, HOMES, RFT l, 1L2R.B, HCST, ONLY, .LRD1 ,

PLEKHFL ΡΥΗΪΝ1 , PTPN4, MATK, STAT4, ADRB2, ARHGEF3. CCL4.L2, GNLY, ST 39„ HOPX, MAP4KL ZAP70, SKAPl , SH2D1A, YWHAO, CD8A„ PR CQ, CX3CRI , LOC729495, IL.10RA, GZMB, CX3CR1 , CDC25B, RALGDS, RAP2A, LOC653438, TARP, FLJ20699, KLRG1 , KLRFl, CD8A, FAM108A2, GNLY, SYTL2, MCOLN2, TTC38, KLRFl , GNPTAB, CCL5, SYTI i , HS.19339, CTS W, CBLB,

APOBEC3G, ST6GALL AUTS2, FLI14213, LOC643035, KIR2DL3, KLRC3, KIR2DL4, GPSL ZNF683, PATL2, MYOM2, KD 5B, BCL2, BCL9L, GATA3, NLRP2, ΖΜΪΖ.2, FBX07, VAMP2, ESFl , PAOX. ΪΤΜ2Α, more particularly CYP27B1 CD40, CYBB, ILI 5, 1 Fill I . APOBE G, C3, CF.B, NML IDOl, PLA2G7. APOLL TAP.l TAP2, TAPBP, NDSTl, PPBP, CCL8, CCL13, (XL22, CCL24, or CXCLiO, are well known to those skillet! in the art, and include ELiSA (enzyme linked immunosorbent assay), RIA (radioimmunoassay). Western blotting, and immunohistochemisoy. Immunoassays such as

ELISA or RIA, which can be extremely rapid, are more generally preferred. These methods use antibodies, or antibody equivalents, to detect IL-32 or biomarker. Antibody arrays or protein chips can also be employed, see for example U.S. Patent Application Nos:

200300.13208A 1; 20020155493 A 1 , 200300175! 5 and U.S. Pa Nos: 6329,209; 6365,418, herein incorporated by reference in their entirety.

ELiSA and RIA procedures may be conducted such that an IL-32 or biomarker standard is labeled (with a radioisotope such as H or ^" S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase and, together with the itrr!abeiled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, IL-32 or biomarker in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled ami- IL-32 or biomarker antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA- sandwich assay). Other conventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a "one-step" or "two-step" assay. A "one-step" assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A "two-step" assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable,

in certain embodiments, a method for measuring IL-32 or biomarker levels comprises: contacting a biological specimen with an antibody or variant (e.g., fragment) thereof which selectively binds IL-32 or biomarker, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of IL-32 or biomarker. A method may further comprise contacting the specimen with a second antibody, e.g., a labeled antibody. ^" The method may further comprise one or more steps of washing, e.g., to remove one or more reagents.

Enzymatic and radio-labeling of IL-32 or biomarker and/or the antibodies may be effected by any suitable means. Such means ma generally include covafcnt linking of the enz me to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and may only yield a proportion of active enzyme.

it may be desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and rtme-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually

sufficient.

It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which, are well-known in the art. Simple polyethylene may provide a suitable support.

Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

Other techniques ma be used to detect 1L-32 or biomarker according to practitioner's preference based upon the present disclosure. One such technique is Western blotting (Tovvbin et a!,, Pmc. Na Acad Sci. 76:4350 ( 1 79}), wherein a suitably treated sample is ran on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Ann- 11,-32 or biomarker antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including ^{L': '} i, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.

immunohistoehemisrry may be used to detect expression of human IL-32 or biomarker, e.g., in a urine or blood sample, A suitable antibod is brought into contact with, for example, a thin layer of ceils, washed, and then contacted with- a second, labeled antibody. .Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radioiaheiling. The assay is scored visually, using microscopy. The results may be quantitated, e.g., as described in the Examples.

Inumuiolhstoehemical analysis optionally coupicd with quantification of the signal may be conducted as follows. IL-32 and biomarker expression may be directly evaluated inhe tissue by preparing imraunohisiochemica ^' lly stained slides with, e.g., an avidin- btotinylated peroxidase complex system.

Evaluation of the presence of stains, i.e., IL-32 or biomarker, may also be done by quantitative immunohistochemical investigation, e.g., with a computerized imag analyzer (e.g., Automated Cellular Imaging System, AGS, Chroma Vision Medical System Inc., San Juan Capistxano, CA) may be used for evaluation of the ievels of IL-32 or biomarker expression in the immunostained tissue samples. Using ACIS, "cytoplasmic staining" ma ^¬ be chosen as program tor IL-32 or biomarker detection. Different areas of immunostained rumor samples may be analyzed with the AOS system. An average of the ACIS values that is more or less than 1, e.g., about 1,1 , 1,2, 1,3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, 100 or more indicates an elevated or decreased IL-32 or biomarker expression.

Other machine or aiftotmagiog systems may also be used to measure

im unostatning results for IL-32 or biomarker. As used herein, "quantitative"

immunohistoeheroistry refers to an automated method of scanning and scoring samples that have undergone immunohistochemistry, to identify and quantitate the presence of a specified biomarker, such as an antigen or other protein. The score given to the sample is a numerical representation of the intensity of the immunohistoehemical staining of the sample, and represents the amount of target biomarker present in the sample. As used herein. Optical Density (OD) i a numerical score that represents intensity of staining. As used herein, semi-quantitati ve immunohistochemistry refers to scoring of

imminiohistochemical results by human eye, where a trained operator ranks results numerically (e.g., as 1 , 2 or 3).

Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art Such systems may include automated staining (see, e.g. the Benchmark ^{1 ¾¾} system, Ventana Medical Systems, Inc..) and microscopic scanning, computerized image analysis, serial section comparison (to control for variatio in the orientation and size of a sample), digital repori generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on ceiis and tissues, including immunostained samples. See, e.g., the CAS-200 system (Beeton, Dickinson & Co.).

Another method that may be used for detecting and quanritaring 11,-32 or biomarker protein levels is Western blotting, e.g., as described in the Examples. Tumor tissues may be frozen and homogenized in lysis buffer. Immunodetection can be performed with a IL-32 or bioniarker antibody using the enhanced c ^' hemihsmineseeriee system (e.g., from

PerkiriElmer Life Sciences, Boston, MA). The membrane may then be stripped and re- blotted with a control antibody, e.g., anti-aetk (A-2066) poly clonal antibody from Sigma (St. Louis, MO). The intensity of the signal may be quantified by densitometry software (e.g., NIB Image 1.61). After quantification of the IL-32 or biomarker and control signals (e.g., actio), the relative expression levels of IL-32 or biomarker are normalized by amount of die actio in each lane, i.e., the value of the IL-32 or biomarker signal is divided fay the value of the control signal, IL-32 or biomarker protein expression is considered to be elevated when the relative level is more than 1 , e.g., about 1.1, 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, 100. Conversely, IL-32 or biomarker is considered to be reduced when the relative level is less than 1, e.g., about 1.1 , 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, 100.

AntML-32 or biomarker antibodies may also be used for imaging purposes, for example, to detect the presence of IL-32 or biomarkers in cells and tissues of a subject. Suitable labels include radioisotopes, iodine ( ^{! * !} 1), carbon ( ^l C)„ sulphur ( ^; S), tritium CH), indium ( ^! '"To), and technetium ("mTe), fluorescent labels, such as fluorescein and rhodamine, and biotin. immunoenzymatie interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different, chroraogetis such as DAB, AEC or Fast Red,

For Hi vivo imaging purposes, antibodies are not intrinsically detectable from outside the body, and so must be labeled, or otherwise modified, to permit detection.

Markers for this purpose may be any that do not substantial iy interfere with the antibody binding, but which allow external detection. Suitable markers may iticiude those that may be detected by X-radiography, NM or MRI. For X-radiographic techniques, suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the patient, such as barium or caesium, for example. Suitable markers for NMR and MRI generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant- hybridoma, For example.

The size of the subject, and the imaging system used, may determine the quantity of imaging moiety needed to produce diagnostic images, In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected may normally range from about 5 to 20 millicuries of technetium- 9m. The labeled antibody or antibody fragment may then preferentially accumulate at the location of cells which contain IL-32 or biomarker. The !abeied antibody or variant thereof e.g., antibody fragment, can then be detected using known techniques.

Antibodies that may be used to detect IL-32 or biomarker include any antibody, whether natural or synthetic, foil length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the IL-32 or biomarker to be detected. An antibody may have a Kd of at most about 10 ^"6 M, ΜΓΜ, 10 ^"S M ₅ .! ⁽ } ^" ¼ 10 ^{' i0} M, 10 ^"H M, 10 ^* M. The phrase "specifically binds" refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant An antibody may bind preferentially to IL-32 or biomarker relative to other proteins.

Antibodies and derivatives thereof that may be used encompasses polyclonal or monoclonal antibodies, chimeric, human, humanized, priraatized (CDR-grafted} _; veneered or single-chain antibodies, phase produced antibodies (e.g., from phage display libraries), as well as functional, i.e., IL-32 or biomarker binding fragments, of antibodies. For example, antibody fragments capable of binding to IL-32 or portions thereof including, but not limited to Fv, Fab, Fab' and F(ab'>2 fragments can be used. Such fragments can be

produced by enzymatic cleavage or by recombinant techniques. ^' For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or Fiab'}? fragments. Antibodies can al o be produced in a variet of truncated form using antibody genes in whteh one or more stop eodons have been introduced itpstream of the natural stop site. For example, a chimeric gene encoding a F{ab¾ heavy chain portion can be designed to include D A sequences encoding the CH, domain and hinge region of the heavy chain.

in some embodiments, agents that specifieaily bind to IL-32 or biomarker or other than antibodies are used, such as peptides. Peptides that specifically bind to IL-32 or biomarker can be identified by any means known in the art. For example, specific pepride binders of IL-32 or biomarker can be screened for using peptide phage display libraries.

Generally, a reagent that is capable of detecting an IL-32 or biomarker polypeptide, such that the presence of IL-32 or biomarker is delected and or quantitated, may be used . A defined herein, a "reagent" refers to a substance that- is cababie of identifying or detecting IL-32 or biomarker in a biological sample (e.g., identifies or defects IL-32 or biomarker rnRNA, DMA, and protein). In some embodiments, the reagent is a labeled or !abelable antibody which specifically binds to IL-32 or biomarker polypeptide. As used herein, the phrase "labeled or labelable" refers to the attachin or including of a label (e.g., a marker or indicator) or ability to attach or include a label (e.g., a marker or indicator). Markers or indicators include, but are not limited to, for example, radioactive molecules, colorimetric molecules, and enzymatic molecules which produce detectable changes in a substrate.

In addition, an IL-32 or biomarker protein may be detected using Mass

Spectrometry such as MALDi/TOF (time-of-flight), SELD1/FOF, liquid chromatography- ttiass spectrometry (LC-MS), gas chromatography-raass spectrometry (GC-MS), high performance liquid clHomatography-mass spectrometry (HFLC-MS), capillary

electrophoresis-iiiass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESl-MS MS etc.). See for example, U.S. Patent Application Nos: 20030.1 9001 , 20030134304, 20030077616, which are herein incorporated by reference.

Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecuies, such as proteins (see, e.g., Li et al, (2000) Tibtech 18; 151-160; Rowley et al, (2000) Methods 20: 383-397; and uster and Mann (1998) Curr, Opin. Structural Biol. 8: 393-400). Further, mass specrrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al.. Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88: 133-44 (2000),

In certain embodiments, a gas phase ion spectrophotometer is used, in other embodiments, iaser-desorption/iomzation mass spectrometry is used to analyze the sample. Modem laser desorptiojv'iotuzation mass spectrometry ("LDl-MS") can be practiced in two main variations: matrix, assisted laser desorpti on/ion ization ("MALD1") mass spectrometry and surface-enhanced laser desoi tioa ionization ("SELDI"), In MA.LDI, the anaiyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological molecules without significantly fragmenting them. However, MAL I. has limitations as an analytical tool. It doe not provide means for fractionating the sample, and the matrix material can interfere with detection, especially for low molecular weight ana!ytes. See, e.g., U.S. Pat. No. 5,1 18,937 (Hillenkamp et a!.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

hi SELD1, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest In another variant, the surface is deri vatized with energy absorbing molecules that are not desorbed when struck with the laser, in another variant, the surface is derivatized with molecules that bind the protein of interest and that contain a photoly ic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied. See, e.g., U.S. Pat. Ho. 5,719,060 (Hittcheos &. Yip) and WO 98/59361 (Hutehens & Yip). The two methods can be combined by, for example, using a SELDi affinity surface to capture an analyte and adding matrix- containing liquid to the captured analyte to provide the energy absorbing material.

For additional information regarding mass spectrometers, see, e.g., Principles of instrumental Analysis, 3rd edition., Skoog, Saunders College Publishing, Philadelphia, 1985; and Kirk-Qthmer Encyclopedia of Chemical Technology, 4.* ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071 094.

Detection of the presence of a marker or other substances may typically involve defection of signal intensity. This, in turn, can reflect the quantity and character of a polypeptide bound to the substrate. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared {e.g., visually, by computer analysis etc), to determine the relative amounts of particular btomolecu!es. Software programs such, as the Biomarker Wizard program (Ciphergen Biesysteros, inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.

Any person skilled in the art understands, any of the components of a mass spectrometer (e.g., desorption source, mass analyzer, detect, etc.) and varied sample preparations can be combined with other suitable components or preparations described herein, or to those known in the art. For example, in some embodiments a control sample, a reference sample, and or one or more test samples may be distinguished by the presence of heavy atoms (e.g., ^K, C), optionally by using isotopicaily differentiated labels linked to the substrate to be detected in an array of samples, thereby permitting multiple samples to be combined and differentiated in the same mass spectrometry run.

In certain preferred embodiments, a laser desorption time-of- flight (TOF) mass spectrometer is used. In laser desorption mass spectrometry, a substrate with a bound marker is introduced into an inlet system. The marker is desorbed and ionized into the gas phase by laser from the ionization soitree. The ions generated arc collected by an ion optic assembly, and then in a time-of- flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a di ffcrent time. Since the time-of- flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge -ratio.

in some embodiments, the relative amounts of one or more biomolecules present in a first or second sample is determined, in part, by executing an. algorithm with a programmable digital computer. The algorithm identifies at least one peak value in the first mass spectrum and the second mass spectrum. The algorithm: then compares the signal strength of the peak value of the first mass spectrum to the signal strength of the peak value of the second mass spectrum of the mass spectrum. The relative signal strengths are an indication of the amount of the biomolecule that is present in the first and second samples. A standard containing a known amount of a biomolecule can be analyzed as the second sample to better quantif the amount of the biomolecule present in the first sample, in certain embodiments, the identity of the biomolecules in the first and second sample can also be determined.

V. I ^' L-32 and biomarker D A and R A level detection techniques

Any method for qualitatively or quantitatively detecting I.L-32 or biomarker RNA, e.g., raRNA, may be used.

Detection of RNA transcripts may be achieved by Northern blotting, for example, wherein a preparation of RNA is run. on a denaturing agarose gel and transferred to a suitable support:, such as activated cellulose, nitrocellulose or glass or nylon membranes. BLadioIabeled cDNA or RNA is then hybridized to the preparation _; washed and analyzed by autoradiography.

Detection of RNA transcripts can further be accomplished using amplification methods. For example, it is within the scope of the present invention to reverse transcribe m ^' RNA into cD A followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap iigase chain reaction (RT-AOLCR.) as described by R. L. Marshall et al, PGR Methods and Applications 4: 80-84 (1994).

in certain embodiments, quantitati e real-time polymerase chain reaction (qRT- PCR) is used to evaluate mRNA levels of IL-32 or biomarker (see Examples). l.L-32 or bioraarker and a control mRN A, e.g., glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) mRNA levels may be quantiiatcd in cancer tissue and adjacent benign tissues, for this, frozen tissues may be cut into 5 micron sections and total RN A may be extracted, e.g., by Qiagen RNeasy Mini Kit (Qiagen, inc., Valencia, CA). A certain amount of RNA, e.g., five hundred nanograms of total RNA, from each tissue may be reversely transcribed by using, e.g., Qiagen Omniscripi RT Kit. Two-step qRT-PCR m y be performed, e.g., with the ABi TaqMan PGR reagent kit (ABi Inc, Foster City, CA), and IL-32 or biomarker primers and GAPDH primers, and the probes for both genes on ABI Prism 7700 system. Suitable primers that may be used are set forth in the Examples. The IL-32 or biomarker copy number may then be divided by the GAPDH copy number and multiplied by 1 ,000 to gi ve a value for the particular subject. In other words, the amount of IL-32 or biomarker mRNA was normalized with the amount of GAPDH mRNA measured in the same RNA extraction to obtain a IL-32 or biomarker/GAPDH ratio, A ratio mat is equal to or mote than 1, e.g., about l .l , 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, 100 may be considered as a high IL-32 or biomarker expression.

Other known amplification methods which can be utilized herein include but are not limited to the so-called "NASBA" or "3SR" technique described in PNAS USA 87: 1874- 1878 ( 1990) and also described in Nature 350 (No. 63 13): 91 -92 ( 1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et at, Clin, Chem. 42: 9-13 (1 96) and European Patent Application No. 684315; and target mediated amplification, as described by PCT Publication W09322461. Primers that may be used for amplification of ^* II -32 nucleic acid portions are set forth in the Examples.

In silu hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples may be stained with haematoxyiin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenm may also be used.

Another method for evaluation of IL-32 or biomarker expression is to detect gene amplification by fluorescent in situ hybridization (FISH). FISH is a technique that can directly identify a specific region of DNA or RNA in a cell and therefore enables visual determination of the IL-32 or biomarker expression in tissue samples. The FISH method has the advantages of a more objective scoring system and the presence of a built-in interna! control consisting of the IL-32 or biomarker gene signals present in all nonneoplastic cells in the same sample. Fluorescence in situ hy bridization is a direct in sittt technique that: is relatively rapid and sensitive. FISH test also can be automated.

Inimunohistochemistry can be combined with a FISH method when the expression level of IL-32 or biomarker is difficult to determine by immnnohistochemistry alone.

Alternatively, mRNA expression can he detected on a DNA array, chip or a microarray. Oligonucleotides corresponding to the IL-32 or biomarker may be immobilized on a. chip which is then hybridized with labeled nucleic acids of a test sample obtained from a patient. Positive hybridisation signal can be obtained with the sample containing IL-32 or biomarker transcripts. Methods of preparing DNA arrays and their use are well known in the art (See, for example U.S. Pat. Nos: 6,6 Ϊ 8,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Sehena et al. 1995 Science 20:467-470; Gerhold et al. 1 99 Trends in Biochem. Sci. 24, 168- 1 73; and Lennon et al. 2000 Drug discovery Today 5; 59-65, which are herein incorporated by reference in their entirety). Serial Analysis of Gene Expression (SAGE) can also be performed (See for example U.S. Patent Application 20030215858.).

To monitor mRNA levels, for example, mRN A can be extracted from the biological sample to be tested, reverse transcribed, and fluorescent-labeled eDNA probes are generated. The microarrays capable of hybridizing to IL-32 or biomarker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.

Types of probes for detection of IL-32 or biomarker RNA include cDNA,

riboprebes, synthetic oligonucleotides and genomic probes. The type of probe used may generally be dictated by the particular situation, such as riboprobes for « situ hybridization, and cDNA for Northern blotting, for example. Most preferably, the probe is directed to nucleotide regions unique to IL-32 or biomarker RNA. The probes may be as short as is required to differentially recognize IL-32 or biomarker mRNA transcripts, and may be as short as, for example, 15 bases; however, probes of at least .17 bases, more preferably 1.8 bases and stiii more preferably 20 bases are preferred. Preferably, the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the IL-32 or biomarker gene. As herein used, the term "stringent conditions" means hybridization may occur only if there is at least 95% and preferably at least 97% identity between the sequences.

The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, ^J~ P and - ^,5 S. Labeling with radioisotope may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases.

VI. Methods for treating ML tuberculosis infection

The invention provides methods for preventing, mitigating, treating, or ameliorating a M. tuberculosis infection in patients having a low level of IL-32, with an agent, such as a vaccine, that boosts the level of IL-32. The method further comprises measuring the post- treatment IL-32 level, and administering additional agent if necessary to further increase the patient's IL-32 level

Dose and dose regimen

The IL-32 vaccine can be administered through any suitable route. In some embodiments, the compositions of the invention are suitable for parenteral administration. These compositions may be administered, for example, intraperitoneaily, intravenously, iiitrarena!!y, or intratheea!ly. In some embodiments, the compositions of the invention are injected intra venously. One of skill in the art would appreciate that a method of administering a therapeutically effective substance formulation or composition of the invention would depend on factors such as the age, weight, and phy sical condition of the patient being treated, and the disease or condition being treated. The skilled worker would. thus, be able to select a method of administration optimal For a patient on a case-by-case basis.

Administration of medicament may be indicated for the treatment of mild, moderate or severe acute or chronic symptoms or for prophylactic treatment. t may be appreciated that the precise dose administered may depend on the age and condition, of the patient, the particular particulate medicament used and the frequency of administration and may ultimately be at the discretion of the attendant physician. Typically, administration may occur weekly, though may occur at a regular or irregular frequency, such as daily or monthly or a combination thereof (e.g., daily for five days once a month).

in some embodiments, the total amo unt of a therapeutically effective IL-32 vaccine in a composition to be administered (e.g., injected or intravenously infused) to a patient is one that is suitable for that patient. One of skill in the art would appreciate that different indi viduals may require different total amounts of IL-32 vaccine, in some embodiments, the amount of the IL-32 vaccine is pharmaceutically effective amount. The skilled worker would be able to determine the amount of the IL-32 vaccine i n a composition needed to treat a patient based on factors such as, for example, the age, weight, and physical condition of the patient. The concentration of the IL-32 vaccine depends in part on its solubility in the intravenous administration solution and the volume of fluid that can be administered.

in certain embodiments, an IL-32 vaccine is administered to the subject at a fixed dose ranging from 0.1 mg m ^"1 to 30 mg/m". For example, an IL-32 vaccine may be administered to the subject in a fixed dose of 0.1 nig/nr, 0.5 mg/rn ² , I mg m ² , 3 mg/m ² , 6 mg/m ² , 9 mg/m", 1.2 mg/m ^* , 15 rag/m ² , 18 mg/m', 21 mg/m ^" , 24 mg/m", 27 rag/n , 30 mg/ , 35 mg/nr, 40 mg/m ² , 50 mg/m ^'' , 60 mg, , 70 mg/ttr, 80 irig/tn", 90 mg m ^" , 100 mg m ² , 1 10 mg m' ^' , 120 mg/m'', 130 rog/ro\ 140 g/ro ³ , 150 mg/m", 160 mg/nr ^* , 170 mg/nr, 180 mg/nf, 190 mg/m~, 200 mg/or, etc. Ranges of values between any of the aforementioned recited values are aiso intended to be included in the scope of the invention, e.g., 0.2 mg/rrr\ 0,6 mg/m ^"" , 1.5 mg/m ^? , 2 mg/nr, 4 mg/m ^' \ 8 mg/m ^" , 10 nig/m', 13 mg/nr- , 17 mg/m ^* ', 20 mg/nY ^" , 23 mg m ^"4 , 25 mg/m ^' \ 26 mg/n , 28 mg/nr, 32 mg/nr", 45 mg/nr-, 55 mg/m ² , 65 mg/m", 75 mg/m ² , 85 mg/m~, 95 mg/m ² , 105 mg/m , 1 15 mg/m", 125 mg/m , 135 mg m ² , 145 rag/m", 155 165 mg/nr-, 175 oig/ni", 185 mg/oi ^' , 195 mg nr, 205 mg m ² , as are ranges based on the forementioned doses, e.g., 0.1-5 mg/m ^"' , 5-10 mg/nr, 10- 15 mg/m", 15-20 mg/m ² , 20-25 mg/m ² , 25-30 mg/m", 30-80 mg/m", 80-120 mg m*, 120- 150 mg/n , .150-175 mg m", 175-200 mg/m ^* ,. The total body dose should not exceed 1 g/m ² weekly or 200 mg/m ^" daily times 5

The concentration of die IL-32 vaccine administered can be about 1 ,0 ug/ral, about 2.0 ug/ml, about 3.0 ug/ml, about 4.0 ug/mi, about 5.0 ug/rril, about 6.0 ug/ml, about 7.0 ug/ml, about 8.0 ug/ml, about 9.0 ug/ml, about 10.0 ug mi, about 1 1 .0 ug/mi about .12.0 ug ml, about 13.0 ug/mi, about 14,0 ug/ml, about 15,0 ug/rnl, etc. The IL-32 vaccine can be administered at a dose sufficient to achieve an increase or modulation in the levels of one or more biomarkers, as discussed ^' herein. A patient may be coupled to a monitor that provides continuous, periodic, or occasional measurements during some or all of the course of treatment. The rate of administration may be modulated manually (e.g., by a physician or nurse) or automatically (e.g., by medical device capable of modulating delivery of the composition in response to physiological parameters received from the monitor) to maintain the patient's physiological and/or biomarker parameters within a desired range or above or below a desired threshold,

The IL-32 vaccine may be administered over a period of time selected from at least

8 hours; at least 24 hours; and from 8 hours to 24 hours. The IL-32 vaccine may be administered continuously for at least 2-6 days, such as 2- 1 1 days, continuously for 2-6 days, for 8 hours a day over a period of at least 2-6 days, such as 2-1 1 days. A weaning period (from several hours to several days) may be beneficial after prolonged infusion, in certain embodiments, the duration of treatment may last up to 8 consecutive weeks of dosing or until die development of dose-limiting toxicity,

A 1itimaUhiiMp.®Me.8g t$

The agent to boost IL-32 levels ("IL-32 vaccine"), may be used in the methods of the invention, either alone or in combination with an additional therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art recognized as being useful to treat M. tuberculosis infection, in other embodiments, the additional agent can be 25

hydroxyvttarmn D (25), or another agent, such as a metabolic precursor, that contributes to a patient's levels of 2SLX in amounts sufficient to induce or support a vitamin D-dependerit antimicrobial pathway or an IFN-γ dependent vitamin D pathway. The combination can also include more than one additional agent, e.g., two or three additional therapeutic agents if the combination is such that the formed composition can perform its intended function. IL-32 vaccines may be used in combination with additional therapeutic agents for the treatment of viral, inflammatory, immune diseases, which may act paraiiei to, dependeiii on, or in concert with the !L-32 vaccine.

i ^' ffi c ■' of IL-32 vaccines

The invention aiso provides methods for assessing the effects of the 11,-32 vaccine iii a patient. Such methods ma be used to determine the efficacy of an IL-32 vaccine, or to adjust a patient's dosage in response to the measured effects. Using the methods described herein, the effects of an IL-32 vaccine may be determined or confirmed, and, optionally, used in the method of treating an active tuberculosis infection or preventing a late t M. tuberculosis infection from progressing to an active state.

in certain embodiments, the invention provides a method for determining the efficacy of an I ^' L-32 vaccine, for M. tuberculosis infection in a subject, using the change in IL-32 levels to determine efficacy . In certain embodiments, the efficacy of an IL-32 vaccine is assessed by detecting a change in IL-32 concentration, levels, and/or activity post-treatment, with a reduction in the level of IL-32 being indicative of an undesirable result; whereas an increase in the level of 11,-32 being indicative of a desirable result, in an undersirable result, additional IL-32 vaccine is administered to further increase the patient's IL-32 levels. In other embodiments, the method of preventing, mitigating, treating, or ameliorating a M tuberculosis infection in a patient further comprises measuring the 25.D level pre- reatment, and if the level is low, administering an agent that boosts levels of 25D to the patient. After post -treatment, if the 25 D remains low, additional agent that boosts levels of 25D may be administered to farther increase the patient's 25 D level.

The efficacy of the IL-32 vaccine, alone or in combination with an agent that boosts levels of 25D, may be assessed in con junction with detection of one or more other faiomarkers including, but not limited to, CYP27BL CD40, CYBB, 11.1 , iFIHi ,

APOBEC3G, C3, CFB, NM1, IDO l , PLA2G7, APOLl , TA.PL TAP2, TAPBP, NDSTI, PPBP, CCL8, CCL13, CCL22, CCL24, or CXCL10, for which increased expression, level, concentration, or activity correlates with efficacy of the IL-32 vaccine or 25D treatment. The efficacy may be measured as a statistically significant chnieal response within a patient or patient population over a control patient or patient population.

hi certain embodiments, the levels of at least one biomarker CYP27BI , CD40, CYBB, IL15, IFIBL APOBBC3G, C3, CFB, NMI, IDOl , PLA2G7, APOLl , TAP 1, TAP2, TAPBP, NDST1 , PPBP, CCL8, CCLl 3, CCL22, CCL24, or CXCL10 will increase by 0.1 , 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1,0, Li, 1,2, 1,3, 1,4, 1,5, 1,6, 1,7, 1.8, 1,9, 2, 4, 6. 8, ^" 10- fold in patients given a dose oFIL-32 vaccine, alone or combined with an agent that boosts levels of 25D, relative to the bioraarker level in an untreated patient or a patient treated with placebo.

As can be appreciated from the disclosure above, the present invention has a wide variety of applications. The invention is further illustrated by the following examples, which are only illustrative arid are not intended to limit the definition and scope of the invention in any way.

EXEMPLIFICATIONS

Example 1 : Materials and Methods

Recombinant human !Ι,-32γ (200ng/ml), lL- 10 (lOng/rnl), IL-15 (200ng/ml) and M-CSF (50ng nii.) were purchased from R&D Systems (Minneapolis, MM) and used at indicated concentrations. IFN-γ (l,3ng mi) was purchased from BD Biosciences (Franklin Lakes, NJ), 25D3 was purchased (BioMol). VDR antagonist Z 1.59 222 (VAZ) was from Bayer Sehering AG, and used at 10-8 .

TB dataseis:

Unstimulated, whole blood transcriptional dataseis from tuberculosis patients that included latent TB and active TB patients (two sets included healthy endemic controls) were retrieved from the Gene Expression Omnibus (GEO), (Table Si). Patients were classified according to the criteria defined by the authors of each study. Briefly, active TB was defined as culture positive pulmonary TB, latent TB as asymptomatic IGRA+ patients. The Germany ^* 12 cohort originally reported four 1GRA patients classified as healthy controls (6), hut were clearly identified as latent tuberculosis. Normalized data as processed by author was analyzed unless study was normalized to latent TB (8), in which raw data from each patient group was normalized by robust muitichip average ( MA) separately. When individual genes were examined with more than one probe, the probe with the highest intensity was chosen. Table SL Datasets used in study.

? 1¾ n

ter^

%t -

*! ::;::·;Γ:. Ο

Analysis of ^' latent TB signature:

For each dataset, the fold-change for each gene was calculated fay dividing the average latent TB value by the average active TB or healthy control value. Genes were ranked fay fold-change and filtered by a P- value < 0.05, as determined by two-tailed student's West of log-transformed intensity values, and an intensity' cutoff of experiment minus baseline of five for Jliumina platforms and five hundred for Agilent platform:. Genes with multiple probe sets were consolidated using the highest fold change. Top genes by fold-change from latent TB versus active TB or healthy controls were then compared using Venn (54)

WGCNA analysis:

Weighted Gene Co-Expression Network Analysis (WGCNA) was applied to data as previous described, (55). Data was filtered according to mean probe set expression across all samples to yield a target number of probe sets between 15,000 and 20,000. The function "biockwiseModuies()" was used to construct unsigned, weighted correlation networks with a soft thresholding power of 4 for macrophage data and 9 for TB data, based on the edge distribution of each dataset. For each network, modules of similarly expressed genes were constructed using a measure of network iiiterconnectediiess, topological overlap, which is calculated from an adjacency matrix of pairwise correlations of ail genes raised to the soft thresholding power. Module eigengenes were calculated for each module, and correlated to TB disease status (for TB data) or cytokine treatment status (for adherent PBMC data) by taking the correlation of each module eigengene to the expression profiles of each condition. Correlations and corresponding P-values were displayed in a heatmap using the WGCNA command labeled HeatmapO, with ME correlations and a binary matrix representation of sample treatments where a "Ί" corresponded to the time-point and cytokine treatment used for that particular sample, and a "0" corresponded to all other

timepoints/tfcatnients not used. Similarly, eigengenes for all human genes annotated with Gene Ontology term "defense response" or IL32 were correlated to expression profiles of cytokine treatment status. For each module, hub genes, or genes with high module

membership, were identified based on iwramodular connectivity (kME). The xigned ME() function was used to rank genes within each module Gene relationships within a module were visualized using the visANT program and the exportNetworkToVis ANTQ function

(56) .

Cell-type specific gene deconvolution was performed based upon a database of 687 publically available microarray samples of 24 different cell types, as previously reported

(57) . Briefly, to identify genes with cell type-specific expression, a two-sample comparison using a moderated t-test and fold-change for differential expression was made between those samples associated with a given cell type, as compared, to samples of the other 23 cell types.

Monocyte and macrophage cultures:

Whole blood from healthy donors was obtained with informed consent. Adherent peripheral blood mononuclear cells were isolated from healthy human donors as previously described (20). Monocytes were enriched by negative selection by Easy Sep Human Monocyte Enrichment Kit without CD i 6 Depletion (STEMCELE- Technologies) according to manufacturer's protocol and confirmed by flow cytometry to have greater than 90% purity for CD14+- monocytes. Monocyte derived .macrophages (MDMs) were differentiated from CD 14 positively-selected monocytes stimulated with M-CSF 5 .ng ml for four days as previously described (25). Cells were stimulated with indicated serum: 1 % fetal calf serum (PCS) (Omega Scientific) or 10% pooled non-beat inacti vated human serum with 25Ό3 concentrations determined to be 40 ng ml ± i ng/mi (25D3 sufficient) or 14 ng/rai (25D3 insufficient as previously described (25).

Mi£W.Af sy.^XM0.6 j&fi ^~^ks .t§i

Adherent PBMC were stimulated with IL- ! O 1.0 ng/mi, I.L-.15 200 ng/ml (R&D Systems), or IL-4 King/ml (company) in RPMI 1 40 supplemented with 10% fetal-calf serum. Cells were harvested and monocytes puri fied by CD 14 mierobeads (Miltenyi Biotec) for a monocyte purity of at least 90%. Transcripteme was measured by mkroarrays using Asymetrix Human Ul 33 Plus 2,0 array comparing four healthy donors at 6h and 24h after stimulation and normalized, as previously been describee! (20).

Si. tuberculosis infection:

M. iubercu sis R37Rv were cultured and infected into macrophages as previously described (Phil PMiD: 245032 ⁽ B) at Biosafety level 3 facility. Briefly, M. tuberculosis was plated on 7H1 1 agar plates from frozen stocks for three to four weeks of incubation at 37deg, 5% C02, incubator and the solid colonies were picked and placed IX PBS. The bacterial suspension was gently separated with a sonicating water bath (Bran ton 251 ) for 30 s and then centrifuged at 735 X g for 4 min to create a single-eel i suspension and enumerated by absorbance at 600nm was measured using spectrophotometry. MDMs were infected at a multiplicity of infection (MOD of one bacteria per cell overnight, subsequently the ceils were 'vigorously washed three times with fresh RPMi 1 40 media to remo ve extracellular bacteria. M. ttiberctt sis-mfeetcd MDMs were then stimulated as indicated and incubated for four days .

A imicrobiei ^' assay:

M. tuberculosis viability from infected MDMs was assessed by the real-time PCR- based method as previously described (58-60), which compares I6S RNA levels with genomic DNA (1S61 .10) levels as indicator of bacterial viability. Genomic DNA was isolated from the interphase and phenol-chloroform: method using the back-extraction protocol, as described by the manufacturer. Total RNA and genomic DN A was isolated using TRIzol reagent (Life Technologies) via phenol -chloroform extraction from the aqueous phase, or interphase, respecti vely. RNA was further purified and DNase digestion performed using a RNeasy Miniprcp Kit (QIAGE , Valencia, CA). cDNA was synthesized from the total R.N A using the i Script eD A Synthesis kit (Bio-Rad Hercules, CA), according to manufacturer's recommended protocol. The bacterial 16S rRNA and genomic element DNA levels were assessed from the cD A and DNA using qPCR, the relative ! 6S values were calculated using the AAC analysis, with the IS6110 value serving as the "housekeeping gene."

Real time quantitative PCR fqPCR):

RNA from monocytes/macrophages was isolated, cDN A synthesized, and qPC performed for vitamin D pathway genes (CYP2?bL VDR, carheiicidia (CAMP), and

DEFS4 as previously described (21 ) or H37Rv viability elements 16S, IS6110. Primers sequences specific for ΪΙ,-32-γ are IL32gForward GTAA.TGCTCCTCCCTACTTCT,

IL32Reverse AAAATCTTTCTATGGCCTGGT. TLR7Forward

TCACCAGACTGTTGCTATGATGC, TLRTRcverse CAGCCAAAACCCACTCGGT. Reactions used Sybr Green PCR Master Mix (BioRad), normalized to h36 ^' B4, and relative arbitrary units calculated using AACT analysis as described (21).

Rate of CYP2?bi activity was assessed in 1L-32 and ΓΡΝ-γ 48h treated adherent monocytes as previously described (1 8). Briefly, [3HJ-25D3 was added to 106 treated cells in 200 αί serum-free medium then incubated for 5 h at 37 ^¾ C. Vitamin D metabolites then extracted and separated by HPLC and eiution profiles determined by UV absorbance at 264nm.

Primary macrophage siR A tmnsfection:

O TA GETplus siR ^' NA pools targeting IL32 (L-OI 5988-00-0005) and control non- targeting pool (D-001 S 10- 10-05) were purchased from Dharaiaeon. Lipofectamine-siRNA complexes were formed using Ho 2μΙ Hpofectamine 2000 and 20 to 6Qpmol siRNA according to manufacturer ^' s iosimciioos and acceptable levels of cell viability (>90%) as determined by trypan blue exclusion. MDMs were seeded in a 24-welI plate at.4 x 104 cells and each well transfected with iipofecta ine-siRNA complexes for four hours 37*C, 5% €02, then washed three times and placed in fresh RPMI 1640 in 10% FCS for 24 h to recover. Transfected MDMs were then stimulated as indicated for 24h.

Differences in individual gene expression among tuberculosis patients was tested on GraphPad Prism software on the log-transformed intensity values and to have normal distribution by D'Agostino-Pearson omnibus test, except, latent TB data from Germany '12 which had an inadequate sample size (n ~ 4) for normality test. Datasets with three patient groups were analyzed using an unpaired one-way analysis of variance (A OVA) test, except for German ' 12 in which a no.nparametric Kruskal-Wallis one-way analysis of variance was used. Datasets with two patient groups were analyzed using an unpaired student's Nest, Dataser of active TB patient treatment rimecourse was analyzed using a paired Nest. Differences in qPCR data were analyzed on iog plus one transformation of fold-changes compared to media using a paired student's Nest Example 2:

To identify poten tial correlates of protection aga nst tuberculosis, the gene expression profiles were analyzed in peripheral blood of tuberculosis patients by overlapping four different data sets (Table Si) of gene expression in tuberculosis patients and individuals with latent infection [Berry et a! (UK, (5)),, Berry et a! (South Africa, (5)), Maertzdorf et al (Germany, (6)) and CI. Bloom et al (South A frica (SA) (β) ^' \. Two of the four data sets included healthy individuals as well as individuals with latent infection f Berry et al (UK, ($}), Maertzdorf et al (Germany, (6))] (Supplemental Table 1 ). In total, 185 gene expression profiles were analyzed, from tuberculosis patients (n ^;::: 72), individuals with latent tuberculosis (n ^::: 88) and healthy controls (« ^::: 25), Because the gene expression profiles were obtained using different microarray platforms and from different laboratories, a fold-change ranking was used combined with a nonstri.ngent cutotY (fold-change > .1.2, />- value < 0.05), shown to be more reproducible between platforms and laboratories (9). The top S OOO genes by fold-change that were more highly expressed in latent vs. acti ve tuberculosis patients in each of the four data sets were overlapped, identifying 30 common genes (Figure L Panel A). Similarly, the top genes that were upregulated in latent tuberculosis vs. normal controls in each of the two datasets were overlapped, identifying 7 common genes. Hypothesizing that those genes with elevated expression in latent tuberculosis vs. active tuberculosis and healthy controls were likely to be relevant to protection against progression to active disease, these two data comparisons were overlapped and only a. single common gene emerged, ίί.32. Examination of the original microarray data revealed that 1L-32 mRNA levels were greatest in peripheral blood of patients with latent tuberculosis, lower in healthy controls and lowest in active tuberculosis patients (Figure 3 , Panel B),

After this analysis was completed, a fifth independent data set by Kaforou et al

(South Africa and Malawi, (10)) was found in the Gene Expression Omnibus (GEO). Again, IL-32 mRNA expression was significantly greater in the peripheral blood gene expression profiles from latent tuberculosis (n ^:!£ 83) vs. active tuberculosis (n ^: -97) (Figure L Panel C). In addition, l ^' L-32 mRNA expression was lowest in a group of patients categorized as having "other diseases', including pneumonia, .malignancy, and a. variety of other infections in which tuberculosis was a possible differential diagnosis. The IL-32 mRNA expression was also comparatively low in peripheral blood gene expression profiles from patients with sarcoidosis, although the gene signatures of sarcoidosis and active tuberculosis largely overlapped (6) (Fig. 1. Panel E). This was regards as an independent data set that validates the findings from the previous data. Finally, gene expression profiles were obtained, from peripheral blood of healthy human volunteers vaceinaled with attenuated modified vaccinia virus Ankara expressing Mtb antigen 85A (MVA85A) (J I, 12). While there was elevation in i.FN-γ produced b peripheral T cells in vitro . there was no detectable change in IL-32 mR A levels at two or seven days post vaccination (Fig. 2, Panel G). The phase lib clinical trial of this first new vaccine against tuberculosis in over a half century failed to engender any protection against either infection or disease (/J).

One of the data sets included serial blood samples from tuberculosis patients undergoing chemotherapy (n ^::: 29) (¾, in which the investigators found a decreasing

"molecular response" of genes as therapy progressed. Examination of these data revealed that IL-32 mRNA, although lowest in active tuberculosis patients, increased during chemotherapy as early as two weeks, reaching the levels observed in latent tuberculosis infection by six months of treatment (Figure I, Panel D).

Exam le 3:

Using a systems biology approach to identify modules ofhighly interconnected genes, including those connected to l ^' t-32, weighted gene correlation network analysis f WGCNA) was performed in the data set of latent tuberculosis and active tuberculosis, with and without treatment (8). A co-expression network was constructed .from this microarray dataset based on pafrwisc correlations of gene expression and modules ofhighly interconnected genes that have significant correlated co-expression, with each gene derived by hierarchical clustering (Figure 2, Panel A). The genes were additionally partitioned around medoids, the central value of a group of expression profiles computed from the median values of each sample across the group. The WGCNA approach is unbiased by any supervision derived from databases or publications and reduces multiple hypothesis testing (Figure 2, Panel B). Four of the 15 gene module eigengencs, linear combinations of genes that capture a large fraction of the variance ia each module, were significantly enriched in the l atent tuberculosis group. Of these, the tan module eigengene was significantly associated with latent TB (/ ^> ~va!ue ^™ 0.03), and inversely correlated with active tuberculosis (/'- value ^::: 0.002), but became significantly enriched with active tuberculosis after six months of chemotherapy (i -vaiue ^:::: 0. 05). The 'ME tan" module contained 88 probe sets, including the IL-32 gene. Given that these gene expression profiles were obtained from peripheral blood, decon volution of this module was performed using cell-type specific gene signatures to identity the cell types likely to be responsible for the expression of individual genes. A subset, of genes was found to be expressed in resting monocytes and macrophages, consistent with a role for !L-32 in macrophage fimction (Fig. 3, Panel C). Although not expressed in resting monocytes- iL-32 is known to be induced in monocytes macrophages by stimulation, with IFN-y (14), activation, of OD2 by mummy! dipeptic!e (IS) and activation of TLR4 by LPS (16).

To identify a role for IL-32 in macrophage function, primary cultures of human monocytes were stimulated itt vitro with 1L-15 to induce M 1-tike macrophages associated with host defense against mycobacteria (17, IB), or conversely with 11,-10 or IL-4 to induce M2~like macrophages associated with pathogenesis in mycobacterial infection (18-20). Gene expression profiles were obtained from the cytokine-derived macrophages and analyzed by WGCNA to identify modules of highly correlated genes (figure 2, Panels€ and D). The most significantly correlated module eigengene identified with any condition, represented as MEblack, was associated with IL- 15 stimulation at 24 hours (P-valoc ^::: 6 10- 13), and contained 802 probe sets including IL-32. The ability oflL-15 to induce IL-32, as evident in the mieroarray data (Fig. 4, Panel E), was confirmed by PCR using additional primary human monocytes and was comparable to induction by iF -y (Figure 2, Panel E). IF -γ induction of IL-32 was dependent on IE- 15 as its knockdown significantly reduced IFN-y induction of IL-32 by 96% (Figure 2, Panel F), but did not affect a control gene (Fig, 5, Panel D). Analysis of gene ontology terms revealed that the bkck module contained a 'defense response' cluster of 48 genes (FDR- 3.09x10-5). Both IL-32 and the entire 'defense response' cluster of 1706 probe sets correlated most strongly with It- 15 stimulation at 24 hoars as shown in the module-trait relationship diagram (P-value~ 4x10- 1 1 and lvalue- 1 x1 -6, respectively) (Figure 2, Panel. D).

Example 4:

Deeonvolution of the iL~l S induced 'defense response' cluster by cell type was performed since the macrophages we e derived from adherent peripheral blood

mononuclear cells (PBMC). Of the 48 genes in this cluster, 23 were expressed at baseline in 5 the myeloid cell lineage (Figure 3. Panel A). The correlated network of the IL-15 induced defense cluster, in which the 23 myeloid genes were highlighted, showed that IL-32 was one of the most highly connected genes in the module, a " ub gene" (intramodular connectivity, kME=0. 30, Table S3). IL-32 expression correlated with four components of the vitamin D antimicrobial pathway (21-23), CJP27B1 (the vitamin D l -a-hydroxylase), it ⁾ CD40, CYBB and II-/ J (Figure 3, Panel B). IL-32 was connected to additional genes annotated as antimicrobial, including MHC class 1 presentation, chemotaxis and lipid metabolism. In the mieroarray data, CYP27BI was induced by 36.4 fold by IL- 15 and correlated with IL-32 expression with a topological overlap score of 0.70 (Table S2). it is to be noted that while the network indicates correlations, the causal relationships remain to be

15 formally established.

Table S2. Myeloid genes correlated with IL-32 in 1L-15 "Defense Response" tunctionaJ cluster from black module derived from cytokme-treated macrophages. IL-15 induction by fold change over media at 24 h determined by expression array and topological connectivity determine by WGCNA. Only those genes significantly expressed in myeloid cells, as

20 determined by cell-type deeonvolution in Figure 3, Panel A are shown.

Table S2, Myeloid genes correlated with IL-32 m the IL~15 defense response functional cluster

Although the WGCNA analysis of (he macrophage subsets revealed a link, defined by topological correlation,, between IL-32 and CYP2?bl , this analysis does not define the directionality of the relationship. Treatment of adherent monocytes with IL-32 was found sufficient to induce CYP27bI mRNA, at levels comparable with IFN-γ or !L-15 treatment (Figure 4, Panel A, Table S4), as well as conversion of 25D to the bioaetive .1 ,25- dihydroxyviiamin D ί 1.25D) (Figure 4, Panel B). IL-32 induction of CYP27bi was dose- dependent (figure 4, Panel E) The ability of IFN-γ to induce CYP27bI in macrophages was dependent on IL-32, as shown by knockdown of IL-32 (Figure 4, Pane!€}, but not a control gene (Fig. 6), The cognate molecular target of 1,25D, the vitamin D receptor (VDR) was also upregutated in monocytes by treatment with I L-32 (Figure 4, Panel D).

Example 5:

11,-32 was sufficient by itself to induce mRNA expression of the antimicrobial peptides catheiicidm and DEPB4 in monocytes, at levels comparable with stimulation by i.P -γ or 1L- ^' 15 (Figure 5, Panel A), and was dependent on the VDR since addition of the VDR antagonist VAZ (24) completely blocked induction (Figure 5, Panel B). To determine whether IL-32 was sufficient to induce an antimicrobial activity, macrophages were infected with the live virulent M. tube culosis and treated with IL-32. When the viability of the bacilli assessed four days later, IL-32 had induced an antimicrobial activity of 70% when the macrophages were cultured in 25D sufficient serum (Figure 5. Panel C), in contrast, when the macrophages were cultured n 25D insufficient serum, no antimicrobial activity was observed, in addition, when the 25D insufficient, serum was supplemented in vitro with 25D to sufficient levels, the antimicrobial response was restored. In these experiments, a parallel response to IFN-γ was observed, as previously described ( 25), Together, these data, indicate that IL-32 induces CYP27bl and the VDR, as well as the vitamin D-dependent induction of antimicrobial peptides and antimicrobial activity.

REFERENCES

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