Patent application title: TUBERCULOSIS ANTIGENS, IMMUNOGENIC COMPOSITIONS, DIAGNOSTICS AND METHODS RELATED TO THE SAME
Huiling Han (San Diego, CA, US)
Dongxin K. Xu (San Diego, CA, US)
Alan Greener (San Diego, CA, US)
IPC8 Class: AA61K3904FI
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from bacterium (e.g., mycoplasma, anaplasma, etc.)
Publication date: 2012-01-05
Patent application number: 20120003256
Provided herein are Mtb polynucleotides, recombinant Mtb peptides and
polypeptides, immunogenic Mtb antigens, and Mtb antibodies. Immunogenic
compositions are also provided that may be useful as recombinant, subunit
and DNA vaccines. In addition the invention provides kits comprising Mtb
polynucleotides, Mtb polypeptides, Mtb antibodies, and combinations
1. An isolated polynucleotide encoding an antigenic Mtb polypeptide,
wherein the polynucleotide is selected from the group consisting of: (a)
a polynucleotide sequence comprising of any one of SEQ ID NOs: 13-24; (b)
a fragment of the polynucleotide sequence of (a), wherein the fragment
encodes an antigenic peptide epitope; and (c) a polynucleotide that is at
least 90% identical to the full length polynucleotide of (a) or (b),
wherein said polynucleotide encodes an antigenic peptide epitope.
2. The isolated polynucleotide of claim 1, wherein said mammal is a rabbit, human, guinea pig, or mouse.
3. The isolated polynucleotide of claim 1, wherein the antigenic polypeptide is immunogenic.
4. The Mtb polynucleotide of claim 1, further comprising a promoter operably coupled to the Mtb polynucleotide.
5. The Mtb polynucleotide of claim 1, further comprising a terminator operably coupled to the Mtb polynucleotide.
6. An isolated Mtb polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12; (b) a fragment of the polypeptide of (a), wherein said fragment comprises an antigenic epitope; and (c) a polypeptide that is at least 90% identical to the full length polypeptide of (a) or the full length fragment of (b), wherein said polypeptide or fragment comprises an antigenic epitope.
7. The isolated Mtb polypeptide of claim 6, further comprising an affinity tag operably coupled to the amino terminus or the carboxy terminus.
8. The isolated Mtb polypeptide of claim 7, wherein the affinity tag is selected from the group consisting of an HA tag or a His tag.
9. A composition comprising at lest one isolated Mtb polypeptide selected from the group consisting of SEQ ID NOs: 1-12, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.
CROSS REFERENCE TO RELATED APPLICATIONS
 The present application claims priority to U.S. Provisional Application Ser. No. 61/309,273 filed on Mar. 1, 2010 by Han et al. and entitled "TUBERCULOSIS ANTIGENS, IMMUNOGENIC COMPOSITIONS, DIAGNOSTICS AND METHODS RELATED TO THE SAME," the entire disclosure of which is herein incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
 The present application is being filed along with a sequence listing in electronic format. The sequence listing is provided as a file entitled GTSYS.043A.txt, created Mar. 1, 2011 which is 34.8 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
 Mycobacterium tuberculosis is the causative agent of tuberculosis, a chronic infectious disease that kills approximately 1.8 million people per year. It has been estimated that two billion people are infected with M. tuberculosis worldwide, including 10 million new cases of tuberculosis per year. In recent years there has been an unexpected rise in tuberculosis cases.
 In the U.S., tuberculosis continues to be a major problem especially among the homeless, Native Americans, African-Americans, immigrants, and the elderly. Immunocompromised individuals are particularly susceptible to tuberculosis. Of the 88 million, new cases of tuberculosis projected in this decade, approximately 10% are expected to be attributable to HIV infection. The emergence of AIDS has reactivated millions of dormant cases of tuberculosis (Mtb), causing a sharp rise in the number of tuberculosis-associated deaths.
 Tuberculosis is spread through the air from an infected individual to uninfected individuals through the air. For example, TB can be spread when an individual that has untreated pulmonary TB coughs, sneezes, or laughs in the vicinity of an uninfected individual. The pathogenicity and the means by which the disease is spread, illustrate the need for the development of methods to detect and diagnose the presence of Mtb.
 Traditional vaccine technology suffers from the problem that it often produces various degrees of immunogenicity in different hosts. Often, the only reliably immunogenic composition is a pathogenic microorganism. Immunogenic compositions based on pathogenic microorganisms pose substantial risks, however, as the manufacture and administration of the pathogenic organism carries a risk of infection by the very pathogen the vaccine is designed to treat. Recent, well-publicized problems with influenza vaccine production illustrate the difficulties in producing large quantities of conventional vaccines and the precarious state of worldwide vaccine supplies. In light of general health concerns and the growing threat of bioterrorism, there is a need to develop recombinant and subunit vaccines capable of inducing an appropriate immune response in the context of multiple and genetically diverse hosts. This approach requires the identification of a number of specific antigenic polypeptides. One of the most difficult tasks in developing a protective or therapeutic vaccine, be it a recombinant or genetic, subunit or multi-valent vaccine, is the identification of the appropriate antigens that can stimulate the most rapid, sustained and efficacious immune responses against a particular pathogen for protection and/or therapeutic effect. This is especially challenging when the genome of the pathogen is large and screening for immunogenic antigens is tedious.
 The only available vaccine for tuberculosis, BCG, is both unpredictable and highly variable in protective efficacy. Hundreds of millions of children and newborns have been vaccinated with BCG, yet this has not consistently stopped the spread of the disease. Tuberculosis has become one of the fastest spreading infectious diseases in both industrialized and developing countries worldwide. Doubtful efficacy of vaccination has spurred interest in developing effective alternatives to BCG.
 The emergence of multi-drug resistant strains of M. tuberculosis, or "Mtb", has complicated matters further, with some experts predicting a new tuberculosis epidemic. In the U.S. about 14% of M. tuberculosis isolates are resistant to at least one drug, and approximately 3% are resistant to at least two drugs. Some M. tuberculosis strains have been isolated that are resistant to as many as seven drugs in the repertoire of drugs commonly used to combat tuberculosis. Resistant strains make treatment of tuberculosis extremely difficult, leading to a mortality rate of about 90%, which is one of the reasons it has gained priority as a defined CDC--Category C Biodefense organism.
 Presently, diagnostics for Mtb include sputum smears, culturing the organism, and chest X-rays. Serodiagnostic kits currently on the market exhibit poor sensitivity (less than 50%). The kits with 30-50% sensitivity exhibit low specificity; kits with higher specificity have very low sensitivity (less than 20%). Another method for diagnosing Mtb is based on the Interferon Gamma Release Assay (IGRA) and marketed as QUANTIFERON GOLD®. This method is cumbersome in that fresh blood is necessary and it is used to detect the presence of the organism, not active infection. In addition, it has poor selectivity with HVI positive and other immunocompromised patients.
 Currently, there are no accurate biomarker assays for Mtb available. Tuberculosis-specific biomarkers are invaluable to help guide treatment of patients that may or may not be responding to the specific antibiotic regimen. As it often takes several weeks for clinicians to know whether the treatment course is effective; a biomarker test would enable health workers to determine the efficacy of treatment in a much shorter time frame.
 In the current age, where diagnosis and treatment of tuberculosis is becoming more challenging and immunosuppressive diseases are more prevalent, new diagnostics and vaccines are essential. Thus, there is a need for developing and commercializing effective and reliable Mtb vaccines. In addition, there is a considerable need for additional diagnostic tests or tests to detect active tuberculosis in the face of other diseases such as HIV.
 The embodiments disclosed herein relate to the identification and discovery of certain peptides and peptide fragments derived from M. tuberculosis that exhibit antigenic activity. Accordingly, embodiments provided herein relate to Mtb polypeptides, Mtb nucleic acids, antibodies that specifically bind to Mtb polypeptides, and compositions and kits comprising Mtb polypeptides, compositions and kits comprising Mtb nucleic acids, and compositions and kits comprising antibodies that specifically bind Mtb polypeptides.
 Some embodiments provide an isolated polynucleotide encoding an antigenic Mtb polypeptide, wherein the polynucleotide is selected from the group consisting of: (a) a polynucleotide sequence comprising of any one of SEQ ID NOs: 13-24; (b) a fragment of the polynucleotide sequence of (a), wherein the fragment encodes an antigenic peptide epitope; and (c) a polynucleotide that is at least 90% identical to the full length polynucleotide of (a) or (b), wherein the polynucleotide encodes an antigenic peptide epitope. In some embodiments, the mammal is a rabbit, human, guinea pig, or mouse. In some embodiments the antigenic polypeptide is immunogenic. In some embodiments, a promoter can be operably coupled to the Mtb polynucleotide. In some embodiments, a terminator can be operably coupled to the Mtb polynucleotide.
 Some embodiments provide an isolated Mtb polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12; (b) a fragment of the polypeptide of (a), wherein said fragment comprises an antigenic epitope; and (c) a polypeptide that is at least 90% identical to the full length polypeptide of (a) or the full length fragment of (b), wherein the polypeptide or fragment comprises an antigenic epitope. In some embodiments, an affinity tag can be operably coupled to the amino terminus or the carboxy terminus. In some embodiments, the affinity tag can be an HA tag and/or a His tag.
 Some embodiments prove a composition that includes at lest one isolated Mtb polypeptide selected from the group consisting of SEQ ID NOs: 1-12, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.
 Some embodiments provide a method of generating an immune response in a mammalian host against Mtb. In some embodiments, the method includes providing to the mammalian host an immunogenic composition that includes at least one nucleic acid or fragment thereof selected from the group consisting of: (a) a polynucleotide sequence comprising of any one of SEQ ID NOs: 13-24; (b) a fragment of the polynucleotide sequence of (a), wherein the fragment encodes an antigenic peptide epitope; and (c) a polynucleotide that is at least 90% identical to the full length polynucleotide of (a) or (b), wherein the polynucleotide encodes an antigenic peptide epitope, or any combination thereof, wherein the nucleic acid encodes and expresses in vivo at least one immunogenic polypeptide. In some embodiments, the immunogenic composition includes an adjuvant.
 Some embodiments provide a method of generating an immune response in a mammalian host against Mtb. The method can include the step of providing to the mammalian host an immunogenic composition that includes at least one Mtb polypeptide or immunogenic fragment thereof, selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12; (b) a fragment of the polypeptide of (a), wherein said fragment comprises an antigenic epitope; and (c) a polypeptide that is at least 90% identical to the full length polypeptide of (a) or the full length fragment of (b), wherein the polypeptide or fragment comprises an antigenic epitope. In some embodiments, the immunogenic composition includes an adjuvant.
 Some embodiments provide kits. In some embodiments, the kit can include an isolated Mtb polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12; (b) a fragment of the polypeptide of (a), wherein said fragment comprises an antigenic epitope; and (c) a polypeptide that is at least 90% identical to the full length polypeptide of (a) or the full length fragment of (b), wherein the polypeptide or fragment comprises an antigenic epitope, or any combination thereof; and an adjuvant. In some embodiments, the kit can include at least two of the Mtb polypeptides.
 Some embodiments provide a method of detecting Mtb in a sample. In some embodiments, the method includes the steps of providing a sample, contacting the sample with at least one antibody that specifically binds an Mtb polypeptide, wherein the Mtb polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12; (b) a fragment of the polypeptide of (a), wherein the fragment comprises an antigenic epitope; and (c) a polypeptide that is at least 90% identical to the full length polypeptide of (a) or the full length fragment of (b), wherein the polypeptide or fragment comprises an antigenic epitope, to generate an antibody-polypeptide complex in the presence a sample comprising Mtb, and detecting the presence of the antibody-polypeptide complex. In some embodiments, the sample can be contacted with a plurality of antibodies that collectively bind to a plurality of Mtb polypeptides or immunogenic fragments thereof.
 Some embodiments provide an isolated antibody that specifically binds an Mtb polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12. In some embodiments, the antibody is a polyclonal antibody. In some embodiments the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric antibody. Some embodiments provide antibody fragments, wherein the antibody fragment specifically binds an Mtb polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-12.
BRIEF DESCRIPTION OF THE DRAWINGS
 The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings.
 FIG. 1 illustrates one method used to generate TAP Expression Fragments.
 FIG. 2 displays a method of amplifying multiple genes using TAP technology, expressing said genes products, then purifying and quantifying the resulting polypeptides.
 FIG. 3 demonstrates how a plurality of polypeptides from a target organism can be assayed to determine each polypeptide's ability to elicit a humoral immune response.
 FIG. 4 demonstrates how a plurality of polypeptides from a target organism can be assayed to determine each polypeptide's ability to elicit a cell-mediated response.
 The embodiments disclosed herein relate to compositions for the detection and identification of Mycobacterium tuberculosis, as well as for the generation of an immune response against M. tuberculosis. In particular, the embodiments disclosed herein relate to, inter alia, antigenic Mycobacterium tuberculosis peptides and antigenic fragments thereof, nucleic acids encoding the antigenic peptides and antigenic fragments thereof, immunogenic compositions including the antigenic polypeptides and/or encoding nucleic acids, methods of using the polypeptides and/or fragments as biomarkers, antibodies to the polypeptides and/or fragments, as well as diagnostic kits for the detection of the peptides and nucleic acids disclosed herein.
 Accordingly, some embodiments relate to polypeptides or antigenic fragments of polypeptides from M. tuberculosis ("Mtb"), including polypeptides represented in SEQ ID NO's: 1-12, or antigenic fragments thereof. Accordingly, some embodiments provide polypeptides that comprise, consist essentially of, or consist of polypeptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, or variants thereof, i.e., Mtb polypeptide variants. "Mtb polypeptide variant" means an Mtb polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length Mtb polypeptide sequence, as disclosed herein (e.g., SEQ ID NOs: 1-12, or variants thereof) or any fragment of a Mtb polypeptide sequence that is antigenic as disclosed herein. Such Mtb polypeptide variants include, for instance, Mtb polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length Mtb amino acid sequence. Ordinarily, an Mtb polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length Mtb polypeptide sequence as disclosed herein (e.g., SEQ ID NO: 1-12) or any other specifically defined fragment of a full-length Mtb polypeptide sequence as disclosed herein. Ordinarily, Mtb variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, or more.
 "Percent (%) amino acid sequence identity" with respect to the Mtb polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific Mtb polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is available as described herein. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
 In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
 where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that programs alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, demonstrated herein is a method to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein to the amino acid sequence designated Mtb, wherein "Mtb" represents the amino acid sequence of a hypothetical Mtb polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "Mtb" polypeptide of interest is being compared, and "X, "Y" and "Z" each represent different hypothetical amino acid residues.
 Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, % amino acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the Mtb polypeptide of interest having a sequence derived from the Mtb polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the Mtb polypeptide of interest is being compared which may be a Mtb variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the Mtb polypeptide of interest. For example, in the statement a polypeptide comprising an the amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B, the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the Mtb polypeptide of interest.
 Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.
 In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
 where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
 Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
 In particular embodiments, conservative substitutions of interest are shown below under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions shown below, or as further described below in reference to amino acid classes, are introduced and the products screened.
TABLE-US-00001 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine
 In some embodiments, the variant polypeptides provided herein have are antigenic, comprising an epitope-bearing portion of an Mtb polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983).
 An "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. In some embodiments, the antigenic epitope can be a conformational epitope. Conformational epitopes comprise discontinuous amino acids that come together in three dimensional conformation and interact with an antibody. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against a protein (see, for example, Sutcliffe et al., Science 219:660 (1983)). Accordingly, antigenic epitope-bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides or antigenic-epitope bearing fragments described herein.
 Antigenic epitope-bearing peptides and polypeptides can contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NOs: 2 or 5. Such epitope-bearing peptides and polypeptides can be produced by fragmenting an Mtb polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for identifying epitopes and producing antibodies from small peptides that comprise an epitope are described, for example, by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons 1997).
 In some embodiments, the antigenic epitope is recognized by an antibody produced by a mammal, such as a mouse, rat, monkey, guinea pig, human, or any other mammal. Preferably, the antigenic epitope is recognized by a human antibody.
 An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
 In some embodiments, the Mtb polypeptides disclosed herein can be operably coupled to an affinity tag, either at the N-terminus, the C-terminus, or at both positions. The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., Methods Enymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2:95 (1991). DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).
 Some embodiments relate to Mtb polynucleotides that encode Mtb polypeptides or Mtb antigenic fragments, as discussed above. Accordingly, some embodiments provide polypeptides that comprise, consist essentially of, or consist of polypeptides of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, or variants thereof, i.e., Mtb polynucleotide variants.
 As used herein, the terms nucleotide and polynucleotide include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3'→P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA. The terms also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
 It will be appreciated that, as used herein, the terms "nucleoside" and "nucleotide" will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with a halogen, an aliphatic group, or are functionalized as ethers, amines, or the like. Other modifications to nucleotides or polynucleotides involve rearranging, appending, substituting for, or otherwise altering functional groups on the purine or pyrimidine base which form hydrogen bonds to a respective complementary pyrimidine or purine. The resultant modified nucleotide or polynucleotide may form a base pair with other such modified nucleotidic units but not with A, T, C, G or U. For example, guanosine (2-amino-6-oxy-9-beta.-D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine. Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine may be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine may be prepared by the method of Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides may be prepared using the method described by Switzer et al. (1993), supra, and Mantsch et al. (1993) Biochem. 14:5593-5601, or by the method described U.S. Pat. No. 5,780,610 to Collins et al. The non-natural base pairs referred to as κ and π., may be synthesized by the method described in Piccirilli et al. (1990) Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo[4,3]-pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotidic units which form unique base pairs have been described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra, or will be apparent to those of ordinary skill in the art.
 "Mtb variant polynucleotide" or "Mtb variant nucleic acid sequence" means a nucleic acid molecule which encodes an Mtb polypeptide as defined herein and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length Mtb polypeptide or Mtb polypeptide fragment sequence as disclosed herein. Ordinarily, an Mtb variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length Mtb polypeptide sequence as disclosed herein or any other fragment of a full-length Mtb polypeptide sequence as disclosed herein.
 Ordinarily, Mtb variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
 "Percent (%) nucleic acid sequence identity" with respect to Mtb-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the Mtb nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
 In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
 where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "Mtb DNA" wherein "Mtb-DNA" represents a hypothetical Mtb-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "Mtb-DNA" nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent different hypothetical nucleotides.
 Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, % nucleic acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the Mtb polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence Mtb polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the Mtb polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant Mtb polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the Mtb polypeptide-encoding nucleic acid molecule of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B, the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the Mtb polypeptide-encoding nucleic acid molecule of interest.
 Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.
 In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
 where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
 Variations in the sequence of the polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the native sequence polypeptide.
 In other embodiments, Mtb variant polynucleotides are nucleic acid molecules that encode an active Mtb polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length Mtb polypeptide as disclosed herein. Mtb variant polypeptides may be those that are encoded by an Mtb variant polynucleotide.
 "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
 "Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.
 "Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
 Variant Mtb polynucleotides are generated using any technique known to those skilled in the art, such as saturation mutagenesis, optimized directed evolution, or the like. Gene Site Saturation Mutagenesis®, or "GSSM®." includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, described in detail in U.S. Pat. No. 6,673,552.
 "Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. An "isolated" nucleic acid, such as an isolated Mtb polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
 In some embodiments, the Mtb polynucleotides disclosed herein can be operably coupled to a regulatory element, such as a promoter, a terminator, a ribosome binding site, or the like, as discussed below.
Vectors and Host Cells
 Also provided are expression vectors and cloning vehicles comprising nucleic acids disclosed herein, e.g., sequences encoding Mtb polypeptides, e.g. SEQ ID NOs; 13-24, and variants thereof.
 In some embodiments, the Mtb polynucleotides disclosed herein can be operably coupled to a promoter. As used herein, the term "promoter" refers to a DNA sequence having a regulatory function, which is recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. In some embodiments, the promoter can be derived from a microorganism, such as a bacterial. Exemplary bacterial promoters useful in the embodiments disclosed herein include, but are not limited to. Examples of inducible heterologous promoters used in prokaryotes include bacterial promoters such as the Plac, Ptrp, Ptac promoters.
 Promoters are typically adjacent to the coding sequence of a gene and extend upstream from the transcription initiation site. The promoter regions may contain several short (<10 base pair) sequence elements that bind transcription factors, generally located within the first 100-200 nucleotides upstream of the transcription initiation site. Sequence elements that regulate transcription from greater distances are generally referred to as "enhancers" and may be located several hundred or thousand nucleotides away from the gene they regulate. Promoters and enhancers may be cell- and tissue-specific; they may be developmentally programmed; they may be constitutive or inducible e.g., by hormones, cytokines, antibiotics, or by physiological and metabolic states. For example, the human metallothionein (MT) promoter is upregulated by heavy metal ions and glucocorticoids. Inducible promoters and other elements may be operatively positioned to allow the inducible control or activation of expression of the desired TAP fragment. Examples of such inducible promoters and other regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid-responsive promoters, rapamycin responsive promoters, and the like (see e.g., No, et al., Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al., Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Certain promoters are operative in prokaryotic cells, while different promoter sequences are required for transcription in eukaryotic cells. Additional control elements that can be used include promoters requiring specific transcription factors, such as viral promoters that may require virally encoded factors. Promoters can be selected for incorporation into TAP fragments based on the intended use of the polynucleotide, as one skilled in the art will readily appreciate. For example, if the polynucleotide encodes a polypeptide with potential utility in human cells, then a promoter capable of promoting transcription in mammalian cells can be selected. Typical mammalian promoters include muscle creatine kinase promoter, actin promoter, elongation factor promoter as well as those found in mammalian viruses such as CMV, SV40, RSV, MMV, HIV, and the like. In certain embodiments, it may be advantageous to incorporate a promoter from a plant or a plant pathogen (e.g., cauliflower mosaic virus promoter), a promoter from a fungus such as yeast (e.g., Gal 4 promoter), a promoter from a bacteria or bacterial virus, such as bacteriophage lambda, T3, T7, SP6, and the like.
 The term "terminator" refers to DNA sequences, typically located at the end of a coding region, that cause RNA polymerase to terminate transcription. As used herein, the term "terminator" also encompasses terminal polynucleotide sequences that direct the processing of RNA transcripts prior to translation, such as, for example, polyadenylation signals. Any type of terminator can be used for the methods and compositions of the invention. For example, TAP terminator sequences can be derived from a prokaryote, eukaryote, or a virus, including, but not limited to animal, plant, fungal, insect, bacterial and viral sources. In one embodiment, artificial mammalian transcriptional terminator elements are used. A nonexclusive list of terminator sequences that may be used in the present invention include the SV40 transcription terminator, bovine growth hormone (BGH) terminator, synthetic terminators, rabbit β-globin terminator, and the like. Terminators can also be a consecutive stretch of adenine nucleotides at the 3' end of a TAP fragment.
 Expression vectors disclosed herein may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. The terms "vector" and "expression cassette" as used herein can be used interchangeably and refer to a nucleotide sequence which is capable of affecting expression of a nucleic acid, e.g., a mutated nucleic acid of the invention. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. "Operably linked" as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and includes both the expression and non-expression plasmids.
 The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. A variety of cloning techniques are disclosed in Ausubel et al. Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et at., Molecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press (1989). Such procedures and others are deemed to be within the scope of those skilled in the art. The vector may be, for example, in the form of a plasmid, a viral particle, or a phage. Other vectors include chromosomal, nonchromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus and pseudorabies. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y., (1989).
 Also provided herein are transformed cells that comprise a nucleic acid sequence of the embodiments described herein, e.g., a sequence encoding an Mtb polypeptide or variant described herein, or an expression cassette, e.g., a vector, of the described herein. The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Exemplary bacterial cells include E. coli, Lactococcus lactis, Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium or any species within the genera Bacillus, Streptomyces and Staphylococcus. Exemplary insect cells include Drosophila S2 and Spodoptera Sf9. Exemplary yeast cells include Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Preferably, the host cell is a fungal cell. Exemplary fungal cells include species of Aspergillus, e.g. A. niger, species of Neurospora, e.g., N. crassa, and the like. Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, U.S. Pat. No. 5,750,870. The vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, L, Basic Methods in Molecular Biology, (1986)). In one aspect, the nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, lipofection (e.g., LIPOFECTIN®), electroporation, viral infection, etc. The candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.).
 The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Depending upon the host employed in a recombinant production procedure, the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated. Polypeptides of the invention may or may not also include an initial methionine amino acid residue. Cell-free translation systems can also be employed to produce a polypeptide of the invention. Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof, hi some aspects, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof. The expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. Host cells containing the polynucleotides of interest, e.g., nucleic acids of the invention, can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.
 Also provided are methods for overexpressing recombinant Mtb polypeptides in a cell comprising expressing a vector comprising a nucleic acid disclosed herein, e.g., a nucleic acid comprising a nucleic acid sequence with at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOs: 13-24 over a region of at least about 100 residues, or more, as described above.
 Expression or overexpression of the Mtb polypeptides can be effected by any means, e.g., use of a high activity promoter, a dicistronic vector or by gene amplification of the vector. The polypeptides encoded by the nucleic acids disclosed herein can be expressed, or overexpressed, in any in vitro or in vivo expression system. Any cell culture systems can be employed to express, or over-express, recombinant protein, including bacterial, insect, yeast, fungal or mammalian cultures. Over-expression can be effected by appropriate choice of promoters, enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors (see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun. 229:295-8), media, culture systems and the like. In one aspect, gene amplification using selection markers, e.g., glutamine synthetase (see, e.g., Sanders (1987) Dev. Biol. Stand. 66:55-63), in cell systems are used to overexpress the polypeptides of the invention.
 In preferred embodiments, the Mtb polypeptides and Mtb polynucleotides can be expressed using TAP® technology. With TAP® technology, a particular polynucleotide of interest can be made transcriptionally active and ready for expression in less than one day. "TAP fragments" are transcriptionally active coding sequences prepared using TAP technology, and the two terms can be used interchangeably. TAP fragments encompass polynucleotides that can be readily expressed, for example, by transfection into animal cells or tissues by any nucleic acid transfection technique, without the need for subcloning into expression vectors or purification of plasmid DNA from bacteria. TAP fragments can be synthesized by amplification (e.g., polymerase chain reaction, or PCR) of any polynucleotide of interest using nested oligonucleotide primers. Two polynucleotide sequences are typically incorporated into TAP fragments, one of which comprises an active transcriptional promoter and the other comprises a transcriptional terminator.
 TAP fragments and methods of making the same are described in detail in U.S. Pat. No. 6,280,977, entitled "Method for Generating Transcriptionally Active DNA Fragments" which is hereby incorporated by reference in its entirety. In one embodiment, methods for creating TAP fragments include the steps of: i) designing oligonucleotide primers; ii) amplifying TAP primary fragments; and iii) amplifying TAP expression fragments. FIG. 1 illustrates one method for generating TAP fragments.
 TAP fragments can be prepared using custom oligonucleotide primers designed to amplify a target polynucleotide sequence of interest from the Mtb genome. Primers complementary to the 5' and 3' ends of the polynucleotide of interest can be designed and synthesized using methods well known in the art, and can include any suitable number of nucleotides to permit amplification of the coding region. Typically, the polynucleotide sequence of interest is an open reading frame (ORF) that consists of an uninterrupted stretch of triplet amino acid codons, without stop codons. In certain embodiments, the polynucleotide is a Mtb polypeptide-encoding sequence.
 In one embodiment, 5'-custom oligonucleotide primers of about 41, 42, 43, 44, 45 or 46 nucleotides are designed and synthesized; about 6 nucleotides of which comprise the 5'-TAP end universal sequence 5'-GAAGGAGATATACCATGCATCATCATCATCATCAT-3' (SEQ ID NO: 25) and about 15 to 20 nucleotides are complementary to the Mtb sequence. Accordingly, the target-specific sequence can be, for example, about 15, 16, 17, 18, 19, or 20 nucleotides in length. The 5' oligonucleotide may also incorporate a Kozak consensus sequence (A/GCCAUGG) near an ATG start codon (initiator methionine) for more efficient translation of mRNA. In one embodiment, an ATG start codon is included in the target-specific primer sequence. In another embodiment, an ATG start codon is incorporated into the custom 5'-oligonucleotide when the target sequence encoding a polypeptide of interest lacks an initiation methionine codon at its 5' end
 In one embodiment of the invention, 3'-custom oligonucleotide primers comprise about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides; of these, about 20 nucleotides comprise the 3'-TAP end universal sequence 5'-TGATGATGAGAACCCCCCCC-3' (SEQ ID NO: 26) and about 20 nucleotides are complementary to the target Mtb sequence. In one aspect, a stop codon sequence, can be added to the end of the target Mtb sequence to achieve proper translational termination by incorporating a TCA, TTA, or CTA into the 3'-custom oligonucleotide.
 FIG. 2 illustrates a method for amplifying multiple Mtb polynucleotides using TAP technology, expressing the gene products of the resultant TAP fragments, purifying, and quantifying the resulting polypeptides. FIG. 2 further illustrates a method of preparing polypeptides, which can be assayed to identify their ability to evoke a cell-mediated or humoral immune response.
 Some embodiments provided herein relate to isolated Mtb antibodies, or antibody fragments, that specifically bind Mtb polypeptides. Mtb antibodies can be monoclonal or polyclonal, as discussed further below.
 As used herein, the term "specifically binds" and "specific binding" refers to an antibody (or other molecule), that binds to a target such as an antigen, with greater affinity than it binds to other molecules under the specified conditions of the embodiments disclosed herein. Antibodies or antibody fragments, as known in the art, are polypeptide molecules that contain regions that can bind other molecules, such as antigens. In various embodiments of the invention, "specifically binds" may mean that an antibody or other specificity molecule, binds to a target molecule with at least about a 106-fold greater affinity, preferably at least about a 107-fold greater affinity, more preferably at least about a 108-fold greater affinity, and most preferably at least about a 109-fold greater affinity than it binds molecules unrelated to the target molecule. Typically, specific binding refers to affinities in the range of about 106-fold to about 109-fold greater than non-specific binding. In some embodiments, specific binding may be characterized by affinities greater than 109-fold over non-specific binding. Whenever a range appears herein, as in "1-10 or one to ten, the range refers without limitation to each integer or unit of measure in the given range. Thus, by 1-10 it is meant that each of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any subunit in between.
 Antibodies to a Mtb polypeptide can be obtained, for example, using the product of a Mtb expression vector or Mtb peptide isolated from a natural source as an antigen.
 Anti-Mtb antibodies can be produced using antigenic Mtb epitope-bearing peptides and polypeptides as described herein. Antigenic epitope-bearing peptides and polypeptides of the embodiments disclosed herein contain a sequence of at least four, or between 15 to about 30 amino acids contained within SEQ ID NOs:1-12. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the embodiments disclosed herein, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide or fragment disclosed herein, also are useful for inducing antibodies that bind with Mtb polypeptides. It is desirable that the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
 Potential antigenic sites in Mtb polypeptides, e.g., SEQ ID NOs: 1-12 can be identified using the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default parameters can be used in this analysis.
 The Jameson-Wolf method predicts potential antigenic determinants by combining six major subroutines for protein structural prediction. Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to identify amino acid sequences representing areas of greatest local hydrophilicity (parameter: seven residues averaged). In the second step, Emini's method, Emini et al., J. Virology 55:836 (1985), was used to calculate surface probabilities (parameter: surface decision threshold (0.6)=1). Third, the Karplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to predict backbone chain flexibility (parameter: flexibility threshold (0.2)=1). In the fourth and fifth steps of the analysis, secondary structure predictions were applied to the data using the methods of Chou-Fasman, Chou, "Prediction of Protein Structural Classes from Amino Acid Composition," in Prediction of Protein Structure and the Principles of Protein Conformation, Fasman (ed.), pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; α region threshold=103; β region threshold=105; Garnier-Robson parameters: α and β decision constants=0). In the sixth subroutine, flexibility parameters and hydropathy/solvent accessibility factors can be combined to determine a surface contour value, designated as the "antigenic index." Finally, a peak broadening function can be applied to the antigenic index, which broadens major surface peaks by adding 20, 40, 60, or 80% of the respective peak value to account for additional free energy derived from the mobility of surface regions relative to interior regions. In some embodiments, this calculation is not applied any major peak that resides in a helical region, since helical regions tend to be less flexible.
 Polyclonal antibodies to recombinant Mtb polypeptides or fragments disclosed herein, or to Mtb polypeptides isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995). The immunogenicity of an Mtb polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Mtb polypeptides or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like," such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
 Although polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep, an anti-Mtb antibody of the present invention may also be derived from a subhuman primate antibody. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310 (1990).
 Alternatively, monoclonal anti-Mtb antibodies can be generated. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan"], Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).
 Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a Mtb gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
 In addition, an anti-Mtb antibody may be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
 Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).
 For particular uses, it may be desirable to prepare fragments of anti-Mtb antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
 Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
 For example, Fv fragments comprise an association of VH and VL chains. This association can be noncovalent, as described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992)).
 The Fv fragments may comprise VH and VL chains, which are connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
 As an illustration, a scFV can be obtained by exposing lymphocytes to an Mtb polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Mtb protein or peptide). Genes encoding polypeptides having potential Mtb polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides, which interact with a known target that can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display libraries can be screened using the Mtb sequences disclosed herein to identify proteins which bind to thereto.
 Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995), and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).
 Alternatively, an anti-Mtb antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762 (1997).
 Some embodiments provided herein relate to compositions that comprise, consist essentially of, or consist of one or more antibodies or antibody fragments that specifically bind to an Mtb polypeptide or fragment thereof disclosed herein.
 For example, some embodiments relate to a composition comprising anti-Mtb antibodies disclosed herein, formulated for administration to a subject in need thereof, e.g., for passive immunization as discussed further herein. Other embodiments relate to compositions comprising anti-Mtb antibodies disclosed herein for use as a diagnostic, as disclosed further herein.
Detection of Mtb Polynucleotides and Polypeptides
 Mtb polynucleotides can be used to detect the expression of an Mtb gene in a biological sample. Such probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NOs: 13-25, or a fragment thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:13-24, or a fragment thereof. Probe molecules can be DNA, RNA, oligonucleotides, and the like.
 In a basic detection assay, a single-stranded probe molecule is incubated with isolated nucleic acids from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target species. After separating unbound probe from hybridized molecules, the amount of hybrids is detected.
 Well-established hybridization methods of DNA detection include Southern blot analysis and dot/slot blot hybridization. Nucleic acid probes can be detectably labeled with radioisotopes such as 32P or 35S. Alternatively, Mtb nucleic acids can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Humana Press, Inc. 1993)). Typically, nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
 Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods. Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
 PCR amplification products can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining. Alternatively, fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled Mtb probe, and examined by autoradiography. Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
Detection of Mtb Proteins with Anti-Mtb Antibodies
 Some embodiments relate to the use of anti-Mtb antibodies to screen biological samples in vitro for the presence of Mtb, in order to identify subjects that are infected with Mtb. In one type of in vitro assay, anti-Mtb antibodies are used in liquid phase. For example, the presence of Mtb in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Mtb antibody under conditions that promote binding between Mtb and its antibody. Complexes of Mtb and anti-Mtb antibodies in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or Staphylococcus protein A. The concentration of Mtb in the biological sample will be inversely proportional to the amount of labeled Mtb bound to the antibody and directly related to the amount of free-labeled Mtb. Anti-Mtb antibodies can be used in the same or a similar fashion.
 Alternatively, in vitro assays can be performed in which anti-Mtb antibody is bound to a solid-phase carrier. For example, antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube. Other suitable in vitro assays will be readily apparent to those of skill in the art.
 Immunochemical detection can be performed by contacting a biological sample with an anti-Mtb antibody, and then contacting the biological sample with a detectably labeled molecule that binds to the antibody. For example, the detectably labeled molecule can comprise an antibody moiety that binds to anti-Mtb antibody. Alternatively, the anti-Mtb antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin). Numerous variations of this basic technique are well-known to those of skill in the art.
 Alternatively, an anti-Mtb antibody can be conjugated with a detectable label to form an anti-Mtb immunoconjugate. Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
 The detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the embodiments disclosed herein are 3H, 125I, 131I, 35S and 14C.
 Anti-Mtb immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
 Alternatively, anti-Mtb immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound. The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
 Similarly, a bioluminescent compound can be used to label anti-Mtb immunoconjugates disclosed herien. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
 Alternatively, anti-Mtb immunoconjugates can be detectably labeled by linking an anti-Mtb antibody component to an enzyme. When the anti-Mtb-enzyme conjugate is incubated in the presence of the appropriate substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety, which can be detected, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
 Those of skill in the art will know of other suitable labels, which can be employed in accordance with the embodiments disclosed herein. The binding of marker moieties to anti-Mtb antibodies can be accomplished using standard techniques known to the art. Typical methodology in this regard is described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1 (1977), Shih et al., Intl J. Cancer 46:1101 (1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.
 Moreover, the convenience and versatility of immunochemical detection can be enhanced by using anti-Mtb antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al., "Immunochemical Applications of Avidin-Biotin Technology," in Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).
 Methods for performing immunoassays are well-established. See, for example, Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoassays," in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 180-208, (Cambridge University Press, 1995), Perry, "The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology," in Monoclonal Antibodies: Principles and Applications, Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (Academic Press, Inc. 1996).
 Some embodiments provided herein relate to kits that comprise, consist essentially of, or consist of the Mtb nucleic acids, peptides, and/or antibodies (or any combination thereof), described herein. The kits of the embodiments described herein can be useful for a variety of applications including combining reagents necessary for producing vaccine compositions, as described herein. Such vaccine compositions include the polypeptides and polynucleotides described herein as well as carriers, diluents and other pharmaceutically acceptable carriers. It should be noted, as described above, that the kits may include fragments of the nucleic acids or peptides described herein as well as combinations of the nucleic acids and/or peptides described herein. Preferably the kits include at least 2, 3, 4, 5, 6, 8, 8, 9, 10, 11, 12 or more nucleic acids or peptides described herein. Any combination of the nucleic acids or peptides can be used. In addition, the kits may include adjuvants. In addition, the kits may include instructions for preparing and administering the immunogenic compositions or vaccines.
 In addition, the kits of provided herein find use as diagnostic kits. In particular, the kits find use as serodiagnostic kits. As such, the kits include at least one peptide as described herein. Preferably, however, the kits include a plurality of peptides, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more peptides for diagnosis of Mtb infection or exposure of a subject to Mtb.
 In some embodiments, the nucleic acids encoding the polypeptides find use in diagnostic kits. The nucleic acids encoding the antigenic peptides find use as probes to detect complementary nucleic acids of Mtb. However, in an alternative embodiment the kits include the polypeptides produced from the in vitro transcription-translation reaction find use in detecting antibodies from an organism, animal or patient exposed to Mtb.
 The embodiments disclosed herein also provide kits for performing an immunological diagnostic assay for Mtb gene expression. Such kits comprise at least one container comprising an anti-Mtb antibody, or antibody fragment. A kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Mtb antibody or antibody fragments. Examples of such indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like. A kit may also comprise a means for conveying to the user that Mtb antibodies or antibody fragments are used to detect Mtb polypeptide(s). For example, written instructions may state that the enclosed antibody or antibody fragment can be used to detect Mtb. The written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
 Instructions in the kits disclosed herein can be present in the kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address that may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
 Some embodiments provided herein relate to Mtb immunogenic compositions. "Immunogenic compositions" are preparations that, when administered to a human or non-human animal, elicit a humoral and/or cellular immune response. "Vaccine," as used herein, refers to immunogenic compositions that are administered to a human or non-human patient for the prevention, amelioration or treatment of diseases, typically infectious diseases. "Traditional vaccines" or "whole vaccines" typically may be live, attenuated or killed microorganisms, such as bacteria or viruses. Vaccines also encompass preparations that elicit or stimulate an immune response that may be useful in the prevention, amelioration or treatment of non-infectious diseases. For example, a cancer cell vaccine may be administered to stimulate or supplement a patient's immune response to neoplastic disease. "Subunit vaccines" may be prepared from purified or partially purified proteins or other antigens from a microorganism, cancer cell or other vaccine target. The term "recombinant vaccine" refers to any vaccine that is prepared using recombinant DNA technology and includes certain subunit vaccines (for example, where subunits are cloned and expressed in vitro prior to administration) and "polynucleotide vaccines" such as DNA vaccines that may encode immunogenic polypeptides. Vaccines typically contain at least one immunogenic component (e.g. a cell, virus, polypeptide, polynucleotide, and the like) but may also include additional agents such as adjuvants, which may enhance or stimulate the patient's immune response to the immunogenic component. In certain embodiments, vaccines or components of vaccines may be conjugated e.g. to a polysaccharide or other molecule, to improve stability or immunogenicity of one or more vaccine components.
 In some embodiments, the immunogenic compositions disclosed herein comprise one or more isolated Mtb polypeptide or antigenic-containing epitope fragments thereof, or any combination of Mtb polypeptides or fragments disclosed herein. In some embodiments, the immunogenic compositions comprise, for example, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 Mtb polypeptides (e.g., selected from the group consisting of SEQ ID NO's: 1-12) or antigenic-epitope containing fragments thereof. In some embodiments, the immunogenic composition comprises two or more Mtb polypeptides or antigenic-epitope containing fragments thereof, in the form of a single, recombinant, multivalent polypeptide, as discussed further herein.
 In some embodiments, the immunogenic compositions comprise one or more isolated Mtb polynucleotides or fragments thereof, encoding an Mtb polypeptide or fragment thereof, as described herein, or a combination of Mtb polynucleotides or fragments thereof disclosed herein. In some embodiments the immunogenic compositions comprise, for example, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 Mtb polynucleotides (e.g., selected from the group consisting of SEQ ID NO's: 13-24) or antigenic-epitope encoding fragments thereof. In some embodiments, the immunogenic composition comprises two or more Mtb polynucleotides or antigenic-epitope encoding fragments thereof, operably coupled to encode a single, recombinant, multivalent polypeptide, as discussed further herein.
 In some embodiments, the immunogenic compositions can include a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
 The compositions described herein may contain other ingredients or compounds in addition to Mtb polypeptides and Mtb polynucleotides, including, but not limited to, various other peptides, adjuvants, binding agents, excipients such as stabilizers (to promote long term storage), emulsifiers, thickening agents, salts, preservatives, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. See e.g., U.S. application Ser. No. 09/929,955 and U.S. application Ser. No. 09/930,591. These compositions are suitable for treatment of animals, particularly mammals, either as a preventive measure to avoid a disease or condition or as a therapeutic to treat animals already afflicted with a disease or condition, e.g. M. tuberculosis.
 Many other ingredients can also be present in the immunogenic compositions disclosed herein. For example, Mtb polypeptides and/or polynucleotides disclosed herein can be formulated in admixture with conventional excipients (e.g., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application that do not deleteriously react with the Mtb polypeptides or polynucleotides disclosed herein). Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyetylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Many more suitable carriers are described in Remmington's Pharmaceutical Sciences, 15th Edition, Easton: Mack Publishing Company, pages 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th Edition, Washington, American Pharmaceutical Association (1975).
 The Mtb polynucleotides described herein, in particular, can be formulated with or administered in conjunction with agents that increase uptake and/or expression of the polynucleotides construct by the cells relative to uptake and/or expression of the polynucleotides by the cells that occurs when the identical composition is administered in the absence of such agents. Such agents and the protocols for administering them in conjunction with gene constructs are described in PCT Patent Application Serial Number PCT/US94/00899 filed Jan. 26, 1994. Examples of such agents include: CaPO4, DEAE dextran, anionic lipids; extracellular matrix-active enzymes; saponins; lectins; estrogenic compounds and steroidal hormones; hydroxylated lower alkyls; dimethyl sulfoxide (DMSO); urea; and benzoic acid esters anilides, amidines, urethanes and the hydrochloride salts thereof, such as those of the family of local anesthetics. In addition, the gene constructs can be encapsulated within/administered in conjunction with lipids/polycationic complexes.
 Vaccines can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the adjuvant or the administered nucleic acid or peptide.
 The effective dose and method of administration of a particular vaccine formulation can vary based on the individual patient and the type and stage of the disease, as well as other factors known to those of skill in the art. Therapeutic efficacy and toxicity of the vaccines can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population). The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for human use. The dosage of the vaccines lies preferably within a range of circulating concentrations that include the ED50 with no toxicity. The dosage varies within this range depending upon the type of adjuvant derivative and HCV antigen, the dosage form employed, the sensitivity of the patient, and the route of administration.
 Since many adjuvants have been on the market for several years, many dosage forms and routes of administration are known. All known dosage forms and routes of administration can be provided within the context of the embodiments described herein. Preferably, an amount of adjuvant that is effective to enhance an immune response to an antigen, e.g., an Mtb polypeptide, in an animal can be considered to be an amount that is sufficient to achieve a blood serum level of antigen approximately 0.25-12.5 μg/ml in the animal, preferably, about 2.5 μg/ml. In some embodiments, the amount of adjuvant is determined according to the body weight of the animal to be given the vaccine. Accordingly, the amount of adjuvant in a vaccine formulation can be from about 0.1-6.0 mg/kg body weight. That is, some embodiments have an amount of adjuvant that corresponds to approximately 0.1-1.0 mg/kg, 1.1-2.0 mg/kg, 2.1-3.0 mg/kg, 3.1-4.0 mg/kg, 4.1-5.0 mg/kg, and 5.1-6.0 mg/kg body weight of an animal. More conventionally, the vaccines contain approximately 0.25 mg-2000 mg of adjuvant. That is, some embodiments have approximately 250 μg, 500 μg, 1 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, and 2 g of adjuvant.
 As one of skill in the art will appreciate, the amount of antigens in a vaccine can vary depending on the type of antigen and its immunogenicity. The amount of antigens in the vaccine can vary accordingly. Nevertheless, as a general guide, the vaccines can have approximately 1 μg, 5 μg, 1 μg, 20 μg, 40 μg, 80 μg, 100 μg, 0.25 mg-5 mg, 5-10 mg, 10-100 mg, 100-500 mg, and upwards of 2000 mg of an antigen described herein, for example. Preferably, the amount of antigen is 0.1 μg-1 mg, desirably, 0.1 μg-100 μg, preferably 3 μg-50 μg, and, most preferably, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg-20 μg, when said antigen is a nucleic acid.
 In some approaches described herein, the exact amount of adjuvant and/or antigen is chosen by the individual physician in view of the patient to be treated. Further, the amounts of adjuvant can be added in combination to or separately from the same or equivalent amount of antigen and these amounts can be adjusted during a particular vaccination protocol so as to provide sufficient levels in light of patient-specific or antigen-specific considerations. In this vein, patient-specific and antigen-specific factors that can be taken into account include, but are not limited to, the severity of the disease state of the patient, age, and weight of the patient, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
 Vaccine compositions and immunogenic compositions comprising, consisting of, or consisting essentially of an embodied nucleic acid encoding an Mtb peptide is contemplated. These compositions typically contain an adjuvant, but do not necessarily require an adjuvant. That is, the nucleic acids and peptides described herein can function as immunogens when administered neat. The compositions described herein (e.g., the Mtb immunogens and vaccine compositions containing an adjuvant) can be manufactured in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to animals, e.g., mammals including humans.
 Various nucleic acid-based vaccines are known and it is contemplated that these compositions and approaches to immunotherapy can be augmented by reformulation with an adjuvant. By one approach, for example, a gene encoding one of the Mtb polypeptides described herein is cloned into an expression vector capable of expressing the polypeptide when introduced into a subject. The expression construct is introduced into the subject in a mixture of adjuvant or in conjunction with an adjuvant. For example, the adjuvant is administered shortly after the expression construct at the same site.
 Where the antigen is to be DNA (e.g., preparation of a DNA vaccine composition), suitable promoters include Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine and human metalothionein. Examples of polyadenylation signals useful with some embodiments, especially in the production of a genetic vaccine for humans, include but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal, which is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal.
 More embodiments concern an immunogen comprising the an Mtb polypeptide, or a truncated, mutated, or modified version thereof, as disclosed herein. The immunogen can be provided in a substantially purified form, which means that the immunogen has been rendered substantially free of other proteins, lipids, carbohydrates or other compounds with which it naturally associates.
 Some embodiments contain at least one of the nucleic acids described joined to a support. Preferably, these supports are manufactured so as to create a multimeric agent. These multimeric agents provide the Mtb polypeptide or encoding nucleic acid in such a form or in such a way that a sufficient affinity to the molecule is achieved. A multimeric agent having a Mtb polypeptide or encoding nucleic acid can be obtained by joining the desired molecule to a macromolecular support. A "support" can be a termed a carrier, a protein, a resin, a cell membrane, a capsid or portion thereof, or any macromolecular structure used to join or immobilize such molecules. Solid supports include, but are not limited to, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, animal cells, Duracyte®, artificial cells, and others. An Mtb polypeptide or encoding nucleic acid can also be joined to inorganic carriers, such as silicon oxide material (e.g., silica gel, zeolite, diatomaceous earth or aminated glass) by, for example, a covalent linkage through a hydroxy, carboxy or amino group and a reactive group on the carrier.
 In several multimeric agents, the macromolecular support has a hydrophobic surface that interacts with a portion of the Mtb polypeptide or encoding nucleic acid by a hydrophobic non-covalent interaction. In some cases, the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which hydrophobic groups have been linked such as polystyrene, polyethylene or polyvinyl. Additionally, Mtb polypeptides or encoding nucleic acids can be covalently bound to carriers including proteins and oligo/polysaccharides (e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). In these later multimeric agents, a reactive group on the molecule, such as a hydroxy or an amino group, is used to join to a reactive group on the carrier so as to create the covalent bond. Additional multimeric agents comprise a support that has other reactive groups that are chemically activated so as to attach Mtb polypeptides or encoding nucleic acids. For example, cyanogen bromide activated matrices, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy succinimide chlorformate linkages, or oxirane acrylic supports are used. (Sigma).
 Carriers for use in the body, (i.e. for prophylactic or therapeutic applications) are desirably physiological, non-toxic and preferably, non-immunoresponsive. Suitable carriers for use in the body include poly-L-lysine, poly-D, L-alanine, liposomes, capsids that display the desired Mtb peptide or nucleic acid, and Chromosorb® (Johns-Manville Products, Denver Co.). Ligand conjugated Chromosorb® (Synsorb-Pk) has been tested in humans for the prevention of hemolytic-uremic syndrome and was reported as not presenting adverse reactions. (Armstrong et al. J. Infectious Diseases 171:1042-1045 (1995)). For some embodiments, a "naked" carrier (i.e., lacking an attached Mtb polypeptide or encoding nucleic acid) that has the capacity to attach an Mtb polypeptide or encoding nucleic acid in the body of a organism is administered. By this approach, a "prodrug-type" therapy is envisioned in which the naked carrier is administered separately from the Mtb polypeptide or encoding nucleic acid and, once both are in the body of the organism, the carrier and NS3/4A chimeric polypeptide or encoding nucleic acid are assembled into a multimeric complex.
 The insertion of linkers of an appropriate length between the Mtb or encoding nucleic acid and the support are also contemplated so as to encourage greater flexibility of the Mtb polypeptide, encoding nucleic acid, hybrid, or binding partner and thereby overcome any steric hindrance that can be presented by the support. The determination of an appropriate length of linker that allows for an optimal cellular response or lack thereof, can be determined by screening the Mtb polypeptide or encoding nucleic acid with varying linkers in the assays detailed in the present disclosure.
 A composite support comprising more than one type of Mtb polypeptide or encoding nucleic acid is also envisioned. A "composite support" can be a carrier, a resin, or any macromolecular structure used to attach or immobilize two or more different Mtb polypeptides or encoding nucleic acids. As above, the insertion of linkers, such as λ linkers, of an appropriate length between the Mtb polypeptide or encoding nucleic acid and the support is also contemplated so as to encourage greater flexibility in the molecule and thereby overcome any steric hindrance that can occur. The determination of an appropriate length of linker that allows for an optimal cellular response or lack thereof, can be determined by screening the Mtb polypeptide or encoding nucleic acid with varying linkers in the assays detailed in the present disclosure.
 In other embodiments, the multimeric and composite supports discussed above can have attached multimerized Mtb polypeptides or encoding nucleic acids so as to create a "multimerized-multimeric support" and a "multimerized-composite support", respectively. A multimerized ligand can, for example, be obtained by coupling two or more Mtb polypeptides or encoding nucleic acids in tandem using conventional techniques in molecular biology. The multimerized form of Mtb polypeptides or encoding nucleic acids can be advantageous for many applications because of the ability to obtain an agent with a higher affinity, for example. The incorporation of linkers or spacers, such as flexible λ linkers, between the individual domains that make-up the multimerized agent can also be advantageous for some embodiments. The insertion of λ linkers of an appropriate length between protein binding domains, for example, can encourage greater flexibility in the molecule and can overcome steric hindrance. Similarly, the insertion of linkers between the multimerized Mtb polypeptides or encoding nucleic acids and the support can encourage greater flexibility and limit steric hindrance presented by the support. The determination of an appropriate length of linker can be determined by screening the Mtb polypeptides or encoding nucleic acid in the assays detailed in this disclosure.
 Embodiments also include methods of using vaccine compositions and immunogen preparations comprising the Mtb polypeptides or encoding nucleic acids, or a truncated or mutated version thereof, and, optionally, an adjuvant. The next section describes some of these compositions in greater detail.
Methods of Using the Vaccine Compositions and Immunogen Preparations
 Routes of administration of the embodiments described herein include, but are not limited to, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. Transdermal administration can be accomplished by application of a cream, rinse, gel, etc. capable of allowing the compositions described herein to penetrate the skin. Parenteral routes of administration include, but are not limited to, electrical or direct injection such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal. Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation, either via the mouth or intranasally.
 Compositions that are suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments applied directly to the skin or incorporated into a protective carrier such as a transdermal device ("transdermal patch"). Examples of suitable creams, ointments, etc. can be found, for instance, in the Physician's Desk Reference. Examples of suitable transdermal devices are described, for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen, et al.
 Compositions that are suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline, phosphate buffered saline and oil preparations for injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.
 Compositions that are suitable for transbronchial and transalveolar administration include, but not limited to, various types of aerosols for inhalation. Devices suitable for transbronchial and transalveolar administration of these are also embodiments. Such devices include, but are not limited to, atomizers and vaporizers. Many forms of currently available atomizers and vaporizers can be readily adapted to deliver vaccines having ribavirin and an antigen.
 Compositions that are suitable for gastrointestinal administration include, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration.
 The nucleic acid constructs described herein, in particular, may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectile bombardment gene guns". Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the individual. Such means include, for example, ex vivo transfection, electroporation, microinjection and microprojectile bombardment. After the gene construct is taken up by the cells, they are reimplanted into the individual. It is contemplated that otherwise non-immunogenic cells that have gene constructs incorporated therein can be implanted into the individual even if the vaccinated cells were originally taken from another individual.
 According to some embodiments, the gene construct is administered to an individual using a needleless injection device. According to some embodiments, the gene construct is simultaneously administered to an individual intradermally, subcutaneously and intramuscularly using a needleless injection device. Needleless injection devices are well known and widely available. One having ordinary skill in the art can, following the teachings herein, use needleless injection devices to deliver genetic material to cells of an individual. Needleless injection devices are well suited to deliver genetic material to all tissue. They are particularly useful to deliver genetic material to skin and muscle cells. In some embodiments, a needleless injection device may be used to propel a liquid that contains DNA molecules toward the surface of the individual's skin. The liquid is propelled at a sufficient velocity such that upon impact with the skin the liquid penetrates the surface of the skin, permeates the skin and muscle tissue therebeneath. Thus, the genetic material is simultaneously administered intradermally, subcutaneously and intramuscularly. In some embodiments, a needleless injection device may be used to deliver genetic material to tissue of other organs in order to introduce a nucleic acid molecule to cells of that organ.
 Preferred embodiments include methods of enhancing an immune response to a desired antigen by providing an animal in need with an amount of adjuvant (e.g., ribavirin) and one or more of the nucleic acid or polypeptide compositions disclosed herein that is effective to enhance said immune response. In these embodiments, an animal in need of an enhanced immune response to an antigen/target is identified by using currently available diagnostic testing or clinical evaluation. By one approach, for example, an individual at risk of becoming infected with Mtb, is provided with the vaccine compositions described above in an amount sufficient to elicit a cellular and humoral immune response to Mtb so as to protect said individual from becoming infected with Mycobacterium tuberculosis.
Humoral Immune Response
 Some embodiments relate to the use of the Mtb polypeptide polypeptides disclosed herein, according the methods above (e.g. using TAP or adapter technology) can be used to identify antigenic targets of humoral immunity in Mtb non-human animals and human patients. A humoral immune response relates to the generation of antibodies and their ability to bind to a particular antigen. In general, the humoral immune system uses white blood cells (B-cells), which have the ability to recognize antigens, to generate antibodies that are capable of binding to the antigens.
 In one embodiment, the Mtb polypeptides of the invention are generated according to the methods described above. In certain aspects of this embodiment additional polynucleotide sequences that encode linker molecules are added to the TAP primary fragment or the TAP expression fragment such that the expressed Mtb polypeptides are fused to a linker molecule. As discussed previously, the term "linker molecule" encompasses molecules that are capable of immobilizing the polypeptides to a solid support.
 In a particular embodiment, a Mtb polynucleotide of interest is fused to a HA epitope tag such that the expressed product can include the Mtb gene product fused to the HA epitope. In another embodiment, a Mtb polynucleotide of interest is combined with a histidine (His) coding sequence, such that the expressed product can include the Mtb gene product and a 6×, 7×, 8×, 9×, or 10×histidine tag. In other embodiments a Mtb polynucleotide is combined with a sequence that codes for a GST tag, fluorescent protein tag, or Flag tag. Using these methods it is possible to express and tag every Mtb polypeptide encoded by its genome. In another embodiment, the tagged Mtb polypeptide can be attached to a solid support, such as a 96-well plate. The immobilize polypeptides can be contacted with an antiserum or other fluid containing antibodies from an animal that has been immunized with one or more antigens from Mtb. In one embodiment, ELISA and Western blot assays are performed in parallel to detect the presence of immunogenic Mtb polypeptides.
 As an example of an ELISA assay, tagged Mtb polypeptides can be immobilized on a solid support, such as a 96-well plate. The immobilized Mtb polypeptides are then incubated with serum from an animal that has been immunized with one or more antigens from Mtb, or has been infected directly with Mtb by inoculation, aerosol delivery, or the like. The reaction mixture can be washed to remove any unbound serum antibodies. The ability of the serum antibodies to bind to the bound Mtb polypeptides can then be detected using any one of a number of methods. For example, enzyme linked secondary antibodies can be added to detect the presence of an antigen specific antibody. Any enzyme linked secondary antibody can be used in this invention, depending on the source of the serum. For example, if vaccinated mouse serum is used to provide the primary antibody, enzyme linked anti-mouse antibody can be used as a secondary antibody Likewise if human serum is used to provide the primary antibody, enzyme linked anti-human serum can be used as a secondary enzyme.
 Any suitable assay can be used to determine the amount of bound polypeptide specific antibody. Also, skilled artisans can develop the enzyme assay to determine the amount of polypeptide specific antibody that is bound. In one embodiment, the readout from an assay can show the presence of different levels of antibody in each of the 96 wells. For example, while some Mtb polypeptides are not able to elicit any serum antibodies, other Mtb polypeptides can elicit intermediate levels of antibodies, and some can elicit high antibody levels. In one embodiment, polypeptides that generate high antibody titers can be further researched to determine which polypeptides are present on the surface of the virus. In a particular embodiment of the invention Mtb polypeptides that generate high antibody titers and that are located on the surface of the virus are candidates for use in the development of a subunit Mtb vaccine.
 In addition, serodiagnostic tests may be developed using antigens identified and characterized by these methods. That is, the peptide (epitopes) identified herein find use in detecting antibodies in serum from Mtb infected or exposed organisms, animals or patients.
 FIG. 3 demonstrates one embodiment of determining the humoral immune response generated by an array of polypeptides. One of skill in the art may deviate in certain details from those shown in FIG. 3. For example, the HA tag, or any other tag as described above, may be placed at either the C-terminal or N-terminal end of the polypeptide to insure that epitopes are not concealed due to binding to the plate. Instead of HA tagged polypeptides, a histidine tag can be used, and the polypeptides can be bound to nickel coated plates. For example a 6× 7×, 8×, 9×., or 10× histidine tag can be used. Alternatively, histidine tagged polypeptides can be purified from either transfected cells or from the in vitro transcription translation system. Furthermore, purified Mtb polypeptides can be attached non-specifically to polypeptide-absorbing plates such as Immulon plates, for example.
 In one aspect, highly immunogenic Mtb antigens can be detected by comparing the results of Western blotting analysis with ELISA. Western blotting and ELISA are two independent yet complementary methods that may be used to detect immunogenic Mtb in qualitative and quantitative ways. Western blotting is often used to examine the quality of a polypeptide or protein sample, including such parameters as purity, protein integrity, and degradation. Western blotting detects polypeptides in their denatured form. In one aspect of this embodiment, ELISA, which detects native polypeptides, is used to further examine Western-positive Mtb polypeptides in a more quantitative fashion, to illustrate the strength of the Mtb epitope's immunogenicity.
Cell-Mediated Immune Response
 Mtb peptides disclosed herein can also be exploited to identify the highly immunogenic targets of cell-mediated immunity in Mtb vaccinated non-human animals. In contrast to a humoral immune response, where an antibody binds directly binding to an antigen, a cell-mediated immune response relates to T-cells binding to the surface of other cells that display the antigen. When certain T-cells come into contact with a presented antigen, they produce and release cytokines such as interferon-γ (IFN-γ or Tumor Necrosis Factor-alpha (TNF-.α). Cytokines are cellular signals that can alter the behavior or properties of another cell. For example, cytokines may inhibit viral replication, induce increased expression of MHC class I and peptide transporter molecules in infected cells, or activate macrophages. Accordingly, cytokines released by T-cells, associated with the binding to an antigen, can be used to identify and detect T-cell/antigen interactions.
 Some cells have MHC molecules on their membranes to present antigens to T-cells. Efficient T-cell function relies on proper recognition of the MHC-antigen complex. There are two types of MHC molecules: Class I and Class II. The two different classes of MHC molecules bind peptides from different sources inside the cell for presentation at the cell surface to different classes of T-cells. Any T-cell can be used in the embodiments disclosed herien, and include for example both CD4.sup.+ and CD8.sup.+ T-cells. CD8.sup.+ cells (cytotoxic T-cells) bind epitopes that are part of class I MHC molecules. CD4.sup.+ T-Cells, which includes inflammatory CD4 T-cells and helper CD4 T-cells, bind epitopes that are part of class II MHC molecules. Only specialized antigen-presenting cells express class II molecules.
 There are three main types of antigen-presenting cells: B cells, macrophages and dendritic cells. Each of these cell types is specialized to process and present antigens from different sources to T-cells, and two of them, the macrophages and the B cells, are also the targets of subsequent actions of armed effector T-cells. These three cell types can express the specialized co-stimulatory molecules that enable them to activate naive T-cells, although macrophages and B cells express those molecules only when suitably activated by infection.
 Embodiments relate to detecting Mtb polypeptides capable of evoking a cell-mediated immune response in order to identify potential candidates for use in a subunit vaccine or other pharmaceutical composition. According to one method of detecting a cell-mediated immune response, an Mtb polypeptide is delivered to an antigen-presenting cell where it can be presented in a manner that is recognized by antigen specific T-cells. In another embodiment, a transcriptionally active gene as disclosed herein can be delivered to an antigen-presenting cell where expressed and presented in a manner that can be recognized by antigen specific T-cells. Mtb antigen specific T-cells can be acquired from numerous sources. For example, animals that have been infected, or immunized with one or more antigens from Mtb virus are a good source of antigen specific T-cells. Alternatively, human Mtb patients and volunteers immunized with Mtb can be a source of antigen specific T-cells.
 FIG. 4 demonstrates one embodiment of determining the cell-mediated immune response generated by an array of polypeptides. One of skill in the art may deviate in certain details from those shown in FIG. 4.
 In order to test the ability of Mtb polypeptides to elicit a cell-mediated response, a plurality of Mtb polynucleotides can be amplified and made transcriptionally active using TAP technology. In one embodiment about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 266 Mtb polynucleotides are made transcriptionally active using TAP technology, as described herein.
 In one embodiment, transcriptionally active Mtb polynucleotides can be transfected into an antigen-presenting cell and expressed within the cell. In another embodiment, instead of transfecting the genes into an antigen-presenting cell, the Mtb TAP fragments can be expressed in an in vivo or in vitro (cell-free) expression system and the expressed polypeptide can be delivered into the antigen-presenting cell. The polypeptide can be delivered into the antigen-presenting cell according to any method. In one embodiment, the polypeptide can be delivered using the technology described in U.S. patent application Ser. No. 09/738,046, entitled "Intracellular Protein Delivery Reagent" and U.S. patent application Ser. No. 10/141,535, entitled "Intracellular Protein Delivery Compositions and Methods of Use," both of which are hereby incorporated by reference in their entirety. The reagents described therein are capable of delivering any type of polypeptide into any type of cell.
 In certain embodiments, reagents used to deliver polypeptides into cultured cells can be a cationic lipid formulation. In one embodiment, these reagents can deliver fluorescently labeled antibodies, high and low molecular weight dextrans, phycoerythrin-BSA, caspase 3, caspase 8, granzyme B, and β-galactosidase into the cytoplasm of a variety of different adherent and suspension cells. Caspases delivered to cells with are functional, since they can be shown to send cells into apoptosis. In one embodiment, Mtb polypeptides are delivered into dendritic cells using these reagents.
 Detecting a T-cell's ability to bind to an antigen-presenting cell, after the antigen-presenting cell has processed a particular polypeptide, is useful in determining whether the particular polypeptide evokes a cell-mediated immune response. Once a particular polypeptide is delivered into or expressed in the antigen-presenting cell, an assay can be performed to identify T-cell interaction with the MHC-antigen complex. In one embodiment, it can be determined if T-cells obtained from an animal that was immunized with Mtb can bind to a particular antigen presented by an antigen-presenting cell. For example, an ELIspot assay (Enzyme-Linked Immuno spotting; ELIspot) can be performed to identify antigen specific T-cells. Similar immunoassays can be performed to identify Mtb antigens (presented by an antigen-presenting cells) that stimulate T-cells from active Mtb patients or immunized individuals.
 One method of detecting a T-cell/antigen interaction is to measure the amount of a particular cytokine released by the T-cell when it interacts with a MHC-antigen complex. The skilled artisan can appreciate that other cellular signals can be used to indicate a cell-mediated immune response. In one embodiment, the levels of IFN-γ released by T-cells can indicate whether a particular peptide is capable of evoking a cell-mediated immune response. In a particular embodiment, an antibody specific for IFN-γ can be coated onto a solid support. Unbound antibodies can be washed away and IFN-γ obtained from the supernatant containing T-cells plus antigen-presenting cells or antigen transduced antigen-presenting cells, can be added to the wells. A biotinylated secondary antibody specific for IFN-γ can be added. Excess secondary antibody can be removed and Streptavidin-Peroxidase can be added to the mixture. Streptavidin-Peroxidase is capable of binding to the biotinylated antibody to complete the four-member immunoassay "sandwich." Excess or unbound Streptavidin-Peroxidase is easily removed from the mixture. In order to detect amount of bound Streptavidin-Peroxidase, a substrate solution can be added which reacts with the Streptavidin-Peroxidase to produce color. The intensity of the colored product is directly proportional to the concentration of IFN-γ present in the T-cell/antigen-presenting cell supernatant. Kits for performing these types of immunoassay are readily available from many commercial suppliers or the necessary reagents composing such kits can be purchased separately or produced in-house. In one embodiment, processed and presented Mtb polypeptide that evokes T-cells to produce a high level of IFN-γ can be considered a strong candidate for use in developing a subunit vaccine.
 Those with skill in the art will appreciate that other methods can be used to detect T-cell/Antigen interactions. These methods include bead based assays, flow-based assays, RT-PCR based assays, cytokine ELISAs, lymphoproliferation assays, cytotoxic T cell assays, or any other assay that can detect the interaction of a T-cell with a responder cell (e.g. macrophage).
 The following examples are provided to demonstrate particular situations and settings in which this technology may be applied and are not intended to restrict the scope of the invention and the claims included in this disclosure.
General Procedure for Generating Histidine Tagged TAP Express Fragments
 This example describes the general methodology used to generate a library of M. tuberculosis peptides, which can be used in screening assays described below.
 Briefly, the following procedure was used to produce tagged T7 TAP fragments: Mtb genes are amplified from genomic DNA (Mtb strain H37Rv). A first PCR reaction is run with customized 5' and 3' primers. The 5' primers contain between 43-48 bases. Preferably, the T-7-His TAP ends contain 28 bases while the gene-specific component contained between 15-20 bases. The 3' primers contain between 45-50 bases. Preferably, the T7-terminator TAP ends contained 30 bases while the gene specific component contains between 15-20 bases. The reaction temperature and times for the first PCR reaction are: 94° C. for 2 minutes, followed by 28 cycles of: 94° C. for 20 seconds, 58° C. for 35 seconds, and 70° C. for 2 minutes (for genes that contained more than 2 kb, 1 minute is added for each kb).
 After the first PCR reaction is performed, an aliquot of each PCR reaction from the previous step is transferred into a PCR reaction containing the T7-histidine promoter fragment and T7 terminator fragment. Preferably, the T7 promoter primer contains 25 bases, while the T7-promoter-His tag fragment contains a 104 base EcoRV/BglII fragment. The T7-terminator fragment is a 74 base oligonucleotide. The reaction temperature and times for the second PCR reaction are: 94° C. for 2 minutes, followed by 30 cycles of: 94° C. for 20 seconds, 60° C. for 35 seconds, and 70° C. for 2 minutes (for genes that contained more than 2 kb, 1 minute was added for each kb).
 This reaction was used to generate candidate M. tuberculosis peptides.
Using the Mtb Proteome to Identify the Antigenic Targets of Humoral Immunity in Humans
 The following method was used to systematically screen and identify antigens in Mtb that give rise to a protective humoral immune response in humans. A bioinformatics approach was used to order the M. tuberculosis polynucleotide sequences for amplification. The Mtb genome was first analyzed for hydrophobicity by the method of Doolittle. Hydrophilic polynucleotides sequences were then further grouped by size. Hydrophilic open reading frames/coding regions longer than 500 bp were selected for TAP amplification. Initially, three hundred Mtb genes were synthesized by TAP and ˜3200 proteins were translated and purified in arrays, as described below.
 The PCR reactions were performed such that a nucleotide sequence encoding a 6×His tag was fused to these amplified transcriptionally active genes. The resulting His tagged TAP fragments were expressed to produce ˜3200 Mtb polypeptides containing the His tag.
 A detailed procedure that was used to produce tagged T7-TAP Express fragments is as follows: groups of Mtb polynucleotide sequences were amplified from Mtb genomic DNA. A first PCR reaction was performed using customized 5' and 3' primers. The 5' primers contained between 43-48 bases. In particular, the T-7-His TAP ends contained 28 bases while the gene-specific component contained between 15-20 bases. The 3' primers contained between 45-50 bases. Specifically, the T7-terminator TAP ends contained 30 bases while the gene specific component contained between 15-20 bases.
 The PCR reactions contained 100 ng Mtb genomic DNA, 25 nM final concentration of 5' and 3' primers. Polymerase, PCR buffer and nucleotides were from Clontech. The reaction temperature and times for the first PCR reaction were: 94° C. for 2 minutes, followed by 30 cycles of: 94° C. for 30 seconds, 48° C. for 1 min., and 68° C. for 2.5 minutes.
 Following the first PCR reaction, an aliquot of each PCR reaction containing 100 ng of PCR product from the previous step was transferred into a PCR reaction containing the TAP promoter and terminator fragments. The sequences of these fragments were:
TABLE-US-00002 Promoter fragment: (SEQ ID NO: 27) 5'CGGTCACGCTTGGGACTGCCATAGGCTGGCCCGGTGATGCCG GCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTA ATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACC 3' Terminator fragment: (SEQ ID NO: 28) 5'GGGGGGGGTTCTCATCATCATCATCATCATTAATAAAAGGGC GAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGGCTGCTAACAAA GCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGC ATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAG GAGGAACTATATCCGGAGCGACTCCCACGGCACGTTGGCAAGCTCG 3'
 The reaction temperature and times for the second PCR reaction were: 94° C. for 2 minutes, followed by 30 cycles of: 94° C. for 30 seconds, 48° C. for 60 seconds, and 68° C. for 2.5 minutes.
 The TAP fragments generated by PCR were used as templates for in vitro protein expression using a Roche RTS100 transcription/translation kit according to manufacturer's instructions. Approximately 0.5˜1.0 μg PCR product was used as template, producing approximately 0.5˜5.0 μg of protein per template.
 MAGNEHIS® nickel-coated magnetic beads (Promega) were used to purify the expressed proteins. 15 μl of Ni-magnetic beads (Promega) were pipetted into each well of a microtiter plate. To each well 50 μl wash buffer (50 mM NaHPO4, pH8.0, 300 mM NaCl, 100 mM imidazole) was added with mixing and the plates were placed on a magnetic stand. The supernatant was removed and wash was repeated. 50 μl of the Protein mixture was added with gentle pipetting. The mixture was incubated at room temperature for 2 minutes. The beads were then separated using a magnetic stand, washed 3 times with 150 μl wash buffer and the bound protein was eluted from the beads with 50 μl of 50 mM NaHPO4, pH 8.0, 300 mM NaCl, 250 mM imidazole.
 15 μl of the purified proteins were resolved on 4-12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes The membranes were blocked in TBST/1% BSA, followed by incubation with TBST/1% BSA containing 1000-fold diluted IgG isolated from pooled sera from TB patients. The blots were washed and then incubated with alkaline phosphatase-conjugated goat-anti human serum secondary antibody. Colorimetric development was used to develop the blots. Of the ˜3200 Mtb polypeptides evaluated, 72 were positive in a Western blot using pooled sera from TB patients.
 In order to eliminate Mtb polypeptides from the screen that are present in healthy individuals, Mtb polypeptides that tested positive with pooled IgG isolated from sera from TB patients were tested using a similar Western blot procedure as described above, except that instead of using IgG from pooled sera from TB patients, IgG isolated from pooled sera from healthy individuals was used as the primary antibody. Of the 72 polypeptides that tested positive with sera from TB patients, 53 were also recognized by IgG antibodies from the pooled sera from healthy patients.
 In order to eliminate Mtb polypeptides from the screen that are present in non-tuberculosis mycobacteria, the remaining 19 Mtb polypeptides, recognized by IgG isolated from sera of TB, but not healthy patients, were subjected to a third screen. The remaining 19 polypeptides were tested in a Western blot as described above, except that instead of using IgG from pooled sera from healthy individuals, IgG isolated from pooled sera from individuals infected with non-tuberculosis mycobacteria ("NTM"). Of the 19 Mtb polypeptides tested, 7 were recognized by the IgG isolated from pooled sera from individuals infected with NTM. The remaining 12 Mtb polypeptides are thus useful as antigens/biomarkers.
 The 12 Mtb polypeptides identified in this example are provided as SEQ ID NO's: 1-12. Their encoding nucleic acids are provided herein as SEQ ID NO's: 13-24. This example demonstrates that the Mtb polypeptides and Mtb polynucleotides disclosed herein are useful in immunogenic compositions for prophylactic purposes, i.e., to generate protective immune responses against Mtb, as well as in diagnostic settings.
Using the Mtb Proteome to Identify the Antigenic Targets of Cell-Mediated Immunity in Mtb Vaccinated Mice and Humans
 The following is a method that is used to systematically screen and identify antigens in Mtb that give rise to a protective cell-mediated immune response. Through the use of TAP technology coding sequences of the Mtb genome are amplified. The PCR reactions are performed such that each amplified coding sequence becomes transcriptionally active. The resulting TAP fragments are expressed to produce Mtb polypeptides. Each of the polypeptides is delivered into dendritic cells, located in 96-well plates, using a polypeptide delivery reagent. Serum from Mtb immunized humans is added to each of the different wells.
 An IFN-γ ELIspot assay is run using the following materials and method:
 Millipore 96-well multi-screen filtration plates (Millipore #MAIP S45-10) (Millipore, Bedford, Mass.)
 Anti-IFN-quadrature purified MAb (Clone 1-D1K) (MABTECH #3420-3) (Mabtech, Naka, Sweden)
 Anti-IFN-g Biotinylated MAb (Clone 7-B6-1) (MABTECH #3420-6) (Mabtech, Naka, Sweden)
 Streptavidin-Alkaline Phosphatase (MABTECH #3310-8) (Mabtech, Naka, Sweden)
 Alkaline Phosphate Substrate Kit (BIO-RAD #170-6432) (Bio-Rad, Hercules, Calif.)
 Carbonate Buffer pH 9.6 (0.2 μM sterile filtered)
 RPMI-1640 Medium (GIBCO #22400-089) (Gibco, Grand Island, N.Y.)
 Fetal Bovine Serum (Sigma #F4135-500 mL) (Sigma, St. Louis, Mo.)
 1×PBS (Prepared from 10×PBS DIGENE #3400-1010) (DIGENE, Gaithersburg, Md.)
 Tween® 20 (J. T. Baker #X251-07) (J. T. Baker, Phillipsburg, N.J.)
 96-well plates are coated with Coating Antibody (anti-IFN-g Clone 1-D1K) at 10-15 μg/mL (100 μL/well) and incubated at 4° C. overnight. Using aseptic technique, plates are flicked to remove Coating Antibody and washed 6 times with RPMI-1640. Plates are blocked with 100 μL/well of RPMI-1640+10% FBS (or Human AB serum) for 1-2 hours at room temperature. Plates are flicked to remove blocking buffer and 100 μL/well of antigen specific or control peptides are added at a final concentration of 10 μg/well. Peripheral blood lymphocytes (PBL) are added at 4×105/well and 1×105/well. Plates are incubated at 37° C./5% CO2 for 36 hours. Plates are flicked to remove cells and washed 6 times with PBS+0.05% Tween® 20 at 200-250 μL/well. Plates are blot dried on paper towels.
 Biotinylated antibody (anti-IFN-g Clone 7-B6-1) diluted 1:1,000 in 1×PBS at 100 μL/well is added. The resulting solution is incubated for 3 hours at room temperature. Plates are flicked to remove biotinylated antibody and washed 6 times with PBS+0.05% Tween® 20 at 200-250 μL/well. Plates are blot dried on paper towels. Streptavidin alkaline phosphatase is added at 100 μL/well diluted 1:1,000 in 1×PBS. The plates are incubated for 1 hour at room temperature. Plates are flicked to remove the streptavidin alkaline phosphatase and washed 6 times with 0.05% Tween® 20 at 200-250 μL/well. The plates are washed again 3 times with 1×PBS at 200-250 μL/well. The plates are blot dried on paper towels.
 Substrate is added at 100 μL/well for 10-15 minutes at room temperature. The substrate is prepared according to manufacturer's protocol. The 25× substrate buffer is diluted in dH20 to a 1× concentration. Reagent A & B are each diluted 1:100 in the 1× substrate buffer. Rinsing plates with generous amounts of tap water (flooding plate and flicking several times) stops colorimetric substrate. Plates are allowed to dry overnight at room temperature in the dark. Spots corresponding to IFN-γ producing cells are determined visually using a stereomicroscope (Zeiss KS ELIspot). Results can be expressed as the number of IFN-γ-secreting cells per 106 spleen cells. Responses are considered positive if the response to test Mtb peptide epitope is significantly different (p<0.05) as compared with the response to no peptide and if the stimulation index (SI=response with test peptide/response with control peptide) is greater than 2.0.
Peptide Vaccine Against M. tuberculosis
 An individual at risk of becoming infected with M. tuberculosis is identified using routine clinical methods. The individual is administered an immunogenic composition that includes one or more Mtb polypeptides of SEQ ID NO's: 1-12, or an immunogenic fragment thereof in combination with an adjuvant.
DNA Vaccine Against M. tuberculosis
 An individual at risk of becoming infected with M. tuberculosis is identified using routine clinical methods. The individual is administered an immunogenic composition that includes one or more Mtb polynucleotides as described herein, in an expression system to of SEQ ID NO's: 13-24, or an immunogenic fragment thereof in combination with an adjuvant.
281310PRTMycobacterium tuberculosis 1Met Thr Gly Pro His Pro Glu Thr Glu Ser Ser Gly Asn Arg Gln Ile1 5 10 15Ser Val Ala Glu Leu Leu Ala Arg Gln Gly Val Thr Gly Ala Pro Ala 20 25 30Arg Arg Arg Arg Arg Arg Arg Gly Asp Ser Asp Ala Ile Thr Val Ala 35 40 45Glu Leu Thr Gly Glu Ile Pro Ile Ile Arg Asp Asp His His His Ala 50 55 60Gly Pro Asp Ala His Ala Ser Gln Ser Pro Ala Ala Asn Gly Arg Val65 70 75 80Gln Val Gly Glu Ala Ala Pro Gln Ser Pro Ala Glu Pro Val Ala Glu 85 90 95Gln Val Ala Glu Glu Pro Thr Arg Thr Val Tyr Trp Ser Gln Pro Glu 100 105 110Pro Arg Trp Pro Lys Ser Pro Pro Gln Asp Arg Arg Glu Ser Gly Pro 115 120 125Glu Leu Ser Glu Tyr Pro Arg Pro Leu Arg His Thr His Ser Asp Arg 130 135 140Ala Pro Ala Gly Pro Pro Ser Gly Ala Glu His Met Ser Pro Asp Pro145 150 155 160Val Glu His Tyr Pro Asp Leu Trp Val Asp Val Leu Asp Thr Glu Val 165 170 175Gly Glu Ala Glu Ala Glu Thr Glu Val Arg Glu Ala Gln Pro Gly Arg 180 185 190Gly Glu Arg His Ala Ala Ala Ala Ala Ala Gly Thr Asp Val Glu Gly 195 200 205Asp Gly Ala Ala Glu Ala Arg Val Ala Arg Arg Ala Leu Asp Val Val 210 215 220Pro Thr Leu Trp Arg Gly Ala Leu Val Val Leu Gln Ser Ile Leu Ala225 230 235 240Val Ala Phe Gly Ala Gly Leu Phe Ile Ala Phe Asp Gln Leu Trp Arg 245 250 255Trp Asn Ser Ile Val Ala Leu Val Leu Ser Val Met Val Ile Leu Gly 260 265 270Leu Val Val Ser Val Arg Ala Val Arg Lys Thr Glu Asp Ile Ala Ser 275 280 285Thr Leu Ile Ala Val Ala Val Gly Ala Leu Ile Thr Leu Gly Pro Leu 290 295 300Ala Leu Leu Gln Ser Gly305 3102181PRTMycobacterium tuberculosis 2Val Arg Val Leu Leu Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Gln1 5 10 15Ala Val Lys Leu Ala Glu Lys Leu Gly Ile Pro Gln Ile Ser Thr Gly 20 25 30Glu Leu Phe Arg Arg Asn Ile Glu Glu Gly Thr Lys Leu Gly Val Glu 35 40 45Ala Lys Arg Tyr Leu Asp Ala Gly Asp Leu Val Pro Ser Asp Leu Thr 50 55 60Asn Glu Leu Val Asp Asp Arg Leu Asn Asn Pro Asp Ala Ala Asn Gly65 70 75 80Phe Ile Leu Asp Gly Tyr Pro Arg Ser Val Glu Gln Ala Lys Ala Leu 85 90 95His Glu Met Leu Glu Arg Arg Gly Thr Asp Ile Asp Ala Val Leu Glu 100 105 110Phe Arg Val Ser Glu Glu Val Leu Leu Glu Arg Leu Lys Gly Arg Gly 115 120 125Arg Ala Asp Asp Thr Asp Asp Val Ile Leu Asn Arg Met Lys Val Tyr 130 135 140Arg Asp Glu Thr Ala Pro Leu Leu Glu Tyr Tyr Arg Asp Gln Leu Lys145 150 155 160Thr Val Asp Ala Val Gly Thr Met Asp Glu Val Phe Ala Arg Ala Leu 165 170 175Arg Ala Leu Gly Lys 180350PRTMycobacterium tuberculosis 3Met Asp Phe Val Ile Gln Trp Ser Cys Tyr Leu Leu Ala Phe Leu Gly1 5 10 15Gly Ser Ala Val Ala Trp Val Val Val Thr Leu Ser Ile Lys Arg Ala 20 25 30Ser Arg Asp Glu Gly Ala Ala Glu Ala Pro Ser Ala Ala Glu Thr Gly 35 40 45Ala Gln 504105PRTMycobacterium tuberculosis 4Val Asn Ala Pro Leu Arg Gly Gln Val Tyr Arg Cys Asp Leu Gly Tyr1 5 10 15Gly Ala Lys Pro Trp Leu Ile Val Ser Asn Asn Ala Arg Asn Arg His 20 25 30Thr Ala Asp Val Val Ala Val Arg Leu Thr Thr Thr Arg Arg Thr Ile 35 40 45Pro Thr Trp Val Ala Met Gly Pro Ser Asp Pro Leu Thr Gly Tyr Val 50 55 60Asn Ala Asp Asn Ile Glu Thr Leu Gly Lys Asp Glu Leu Gly Asp Tyr65 70 75 80Leu Gly Glu Val Thr Pro Ala Thr Met Asn Lys Ile Asn Thr Ala Leu 85 90 95Ala Thr Ala Leu Gly Leu Pro Trp Pro 100 1055300PRTMycobacterium tuberculosis 5Met Asp Gln Gln Ser Thr Arg Thr Asp Ile Thr Val Asn Val Asp Gly1 5 10 15Phe Trp Met Leu Gln Ala Leu Leu Asp Ile Arg His Val Ala Pro Glu 20 25 30Leu Arg Cys Arg Pro Tyr Val Ser Thr Asp Ser Asn Asp Trp Leu Asn 35 40 45Glu His Pro Gly Met Ala Val Met Arg Glu Gln Gly Ile Val Val Asn 50 55 60Asp Ala Val Asn Glu Gln Val Ala Ala Arg Met Lys Val Leu Ala Ala65 70 75 80Pro Asp Leu Glu Val Val Ala Leu Leu Ser Arg Gly Lys Leu Leu Tyr 85 90 95Gly Val Ile Asp Asp Glu Asn Gln Pro Pro Gly Ser Arg Asp Ile Pro 100 105 110Asp Asn Glu Phe Arg Val Val Leu Ala Arg Arg Gly Gln His Trp Val 115 120 125Ser Ala Val Arg Val Gly Asn Asp Ile Thr Val Asp Asp Val Thr Val 130 135 140Ser Asp Ser Ala Ser Ile Ala Ala Leu Val Met Asp Gly Leu Glu Ser145 150 155 160Ile His His Ala Asp Pro Ala Ala Ile Asn Ala Val Asn Val Pro Met 165 170 175Glu Glu Met Leu Glu Ala Thr Lys Ser Trp Gln Glu Ser Gly Phe Asn 180 185 190Val Phe Ser Gly Gly Asp Leu Arg Arg Met Gly Ile Ser Ala Ala Thr 195 200 205Val Ala Ala Leu Gly Gln Ala Leu Ser Asp Pro Ala Ala Glu Val Ala 210 215 220Val Tyr Ala Arg Gln Tyr Arg Asp Asp Ala Lys Gly Pro Ser Ala Ser225 230 235 240Val Leu Ser Leu Lys Asp Gly Ser Gly Gly Arg Ile Ala Leu Tyr Gln 245 250 255Gln Ala Arg Thr Ala Gly Ser Gly Glu Ala Trp Leu Ala Ile Cys Pro 260 265 270Ala Thr Pro Gln Leu Val Gln Val Gly Val Lys Thr Val Leu Asp Thr 275 280 285Leu Pro Tyr Gly Glu Trp Lys Thr His Ser Arg Val 290 295 3006228PRTMycobacterium tuberculosis 6Val Arg Ile Lys Ile Phe Met Leu Val Thr Ala Val Val Leu Leu Cys1 5 10 15Cys Ser Gly Val Ala Thr Ala Ala Pro Lys Thr Tyr Cys Glu Glu Leu 20 25 30Lys Gly Thr Asp Thr Gly Gln Ala Cys Gln Ile Gln Met Ser Asp Pro 35 40 45Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser Tyr Tyr Pro Asp Gln Lys 50 55 60Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe Leu Ser Ala65 70 75 80Ala Thr Ser Ser Thr Pro Arg Glu Ala Pro Tyr Glu Leu Asn Ile Thr 85 90 95Ser Ala Thr Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln Ala Val 100 105 110Val Leu Lys Val Tyr Gln Asn Ala Gly Gly Thr His Pro Thr Thr Thr 115 120 125Tyr Lys Ala Phe Asp Trp Asp Gln Ala Tyr Arg Lys Pro Ile Thr Tyr 130 135 140Asp Thr Leu Trp Gln Ala Asp Thr Asp Pro Leu Pro Val Val Phe Pro145 150 155 160Ile Val Gln Gly Glu Leu Ser Lys Gln Thr Gly Gln Gln Val Ser Ile 165 170 175Ala Pro Asn Ala Gly Leu Asp Pro Val Asn Tyr Gln Asn Phe Ala Val 180 185 190Thr Asn Asp Gly Val Ile Phe Phe Phe Asn Pro Gly Glu Leu Leu Pro 195 200 205Glu Ala Ala Gly Pro Thr Gln Val Leu Val Pro Arg Ser Ala Ile Asp 210 215 220Ser Met Leu Ala2257107PRTMycobacterium tuberculosis 7Val Ser Thr Tyr Arg Ser Pro Asp Arg Ala Trp Gln Ala Leu Ala Asp1 5 10 15Gly Thr Arg Arg Ala Ile Val Glu Arg Leu Ala His Gly Pro Leu Ala 20 25 30Val Gly Glu Leu Ala Arg Asp Leu Pro Val Ser Arg Pro Ala Val Ser 35 40 45Gln His Leu Lys Val Leu Lys Thr Ala Arg Leu Val Cys Asp Arg Pro 50 55 60Ala Gly Thr Arg Arg Val Tyr Gln Leu Asp Pro Thr Gly Leu Ala Ala65 70 75 80Leu Arg Thr Asp Leu Asp Arg Phe Trp Thr Arg Ala Leu Thr Gly Tyr 85 90 95Ala Gln Leu Ile Asp Ser Glu Gly Asp Asp Thr 100 1058271PRTMycobacterium tuberculosis 8Met Ala Asn Phe Thr Ala Ala Asp Val Lys Arg Leu Arg Glu Leu Thr1 5 10 15Gly Ala Gly Met Leu Ala Cys Lys Asn Ala Leu Ala Glu Thr Asp Gly 20 25 30Asp Phe Asp Lys Ala Val Glu Ala Leu Arg Ile Lys Gly Ala Lys Asp 35 40 45Val Gly Lys Arg Ala Glu Arg Ala Thr Ala Glu Gly Leu Val Ala Ala 50 55 60Lys Asp Gly Ala Leu Ile Glu Leu Asn Cys Glu Thr Asp Phe Val Ala65 70 75 80Lys Asn Ala Glu Phe Gln Thr Leu Ala Asp Gln Val Val Ala Ala Ala 85 90 95Ala Ala Ala Lys Pro Ala Asp Val Asp Ala Leu Lys Gly Ala Ser Ile 100 105 110Gly Asp Lys Thr Val Glu Gln Ala Ile Ala Glu Leu Ser Ala Lys Ile 115 120 125Gly Glu Lys Leu Glu Leu Arg Arg Val Ala Ile Phe Asp Gly Thr Val 130 135 140Glu Ala Tyr Leu His Arg Arg Ser Ala Asp Leu Pro Pro Ala Val Gly145 150 155 160Val Leu Val Glu Tyr Arg Gly Asp Asp Ala Ala Ala Ala His Ala Val 165 170 175Ala Leu Gln Ile Ala Ala Leu Arg Ala Arg Tyr Leu Ser Arg Asp Asp 180 185 190Val Pro Glu Asp Ile Val Ala Ser Glu Arg Arg Ile Ala Glu Glu Thr 195 200 205Ala Arg Ala Glu Gly Lys Pro Glu Gln Ala Leu Pro Lys Ile Val Glu 210 215 220Gly Arg Leu Asn Gly Phe Phe Lys Asp Ala Val Leu Leu Glu Gln Ala225 230 235 240Ser Val Ser Asp Asn Lys Lys Thr Val Lys Ala Leu Leu Asp Val Ala 245 250 255Gly Val Thr Val Thr Arg Phe Val Arg Phe Glu Val Gly Gln Ala 260 265 2709448PRTMycobacterium tuberculosis 9Leu Thr Gly Pro Glu His Gly Ser Ala Ser Thr Ile Glu Ile Leu Pro1 5 10 15Val Ile Gly Leu Pro Glu Phe Arg Pro Gly Asp Asp Leu Ser Ala Ala 20 25 30Val Ala Ala Ala Ala Pro Trp Leu Arg Asp Gly Asp Val Val Val Val 35 40 45Thr Ser Lys Val Val Ser Lys Cys Glu Gly Arg Leu Val Pro Ala Pro 50 55 60Glu Asp Pro Glu Gln Arg Asp Arg Leu Arg Arg Lys Leu Ile Glu Asp65 70 75 80Glu Ala Val Arg Val Leu Ala Arg Lys Asp Arg Thr Leu Ile Thr Glu 85 90 95Asn Arg Leu Gly Leu Val Gln Ala Ala Ala Gly Val Asp Gly Ser Asn 100 105 110Val Gly Arg Ser Glu Leu Ala Leu Leu Pro Val Asp Pro Asp Ala Ser 115 120 125Ala Ala Thr Leu Arg Ala Gly Leu Arg Glu Arg Leu Gly Val Thr Val 130 135 140Ala Val Val Ile Thr Asp Thr Met Gly Arg Ala Trp Arg Asn Gly Gln145 150 155 160Thr Asp Ala Ala Val Gly Ala Ala Gly Leu Ala Val Leu Arg Asn Tyr 165 170 175Ala Gly Val Arg Asp Pro Tyr Gly Asn Glu Leu Val Val Thr Glu Val 180 185 190Ala Val Ala Asp Glu Ile Ala Ala Ala Ala Asp Leu Val Lys Gly Lys 195 200 205Leu Thr Ala Thr Pro Val Ala Val Val Arg Gly Phe Gly Val Ser Asp 210 215 220Asp Gly Ser Thr Ala Arg Gln Leu Leu Arg Pro Gly Ala Asn Asp Leu225 230 235 240Phe Trp Leu Gly Thr Ala Glu Ala Leu Glu Leu Gly Arg Gln Gln Ala 245 250 255Gln Leu Leu Arg Arg Ser Val Arg Arg Phe Ser Thr Asp Pro Val Pro 260 265 270Gly Asp Leu Val Glu Ala Ala Val Ala Glu Ala Leu Thr Ala Pro Ala 275 280 285Pro His His Thr Arg Pro Thr Arg Phe Val Trp Leu Gln Thr Pro Ala 290 295 300Ile Arg Ala Arg Leu Leu Asp Arg Met Lys Asp Lys Trp Arg Ser Asp305 310 315 320Leu Thr Ser Asp Gly Leu Pro Ala Asp Ala Ile Glu Arg Arg Val Ala 325 330 335Arg Gly Gln Ile Leu Tyr Asp Ala Pro Glu Val Val Ile Pro Met Leu 340 345 350Val Pro Asp Gly Ala His Ser Tyr Pro Asp Ala Ala Arg Thr Asp Ala 355 360 365Glu His Thr Met Phe Thr Val Ala Val Gly Ala Ala Val Gln Ala Leu 370 375 380Leu Val Ala Leu Ala Val Arg Gly Leu Gly Ser Cys Trp Ile Gly Ser385 390 395 400Thr Ile Phe Ala Ala Asp Leu Val Arg Asp Glu Leu Asp Leu Pro Val 405 410 415Asp Trp Glu Pro Leu Gly Ala Ile Ala Ile Gly Tyr Ala Asp Glu Pro 420 425 430Ser Gly Leu Arg Asp Pro Val Pro Ala Ala Asp Leu Leu Ile Leu Lys 435 440 44510392PRTMycobacterium tuberculosis 10Met Ser Arg Ala Phe Ile Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly1 5 10 15Leu Tyr Asp Leu Leu Gly Ile Gly Ile Pro Asn Gln Gly Gly Ile Leu 20 25 30Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala Ala 35 40 45Ala Phe Pro Gly Asp Gly Trp Leu Gly Ser Ala Ala Asp Lys Tyr Ala 50 55 60Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp Leu65 70 75 80Asp Arg Gln Leu Ile Ser Leu Ile His Asp Gln Ala Asn Ala Val Gln 85 90 95Thr Thr Arg Asp Ile Leu Glu Gly Ala Lys Lys Gly Leu Glu Phe Val 100 105 110Arg Pro Val Ala Val Asp Leu Thr Tyr Ile Pro Val Val Gly His Ala 115 120 125Leu Ser Ala Ala Phe Gln Ala Pro Phe Cys Ala Gly Ala Met Ala Val 130 135 140Val Gly Gly Ala Leu Ala Tyr Leu Val Val Lys Thr Leu Ile Asn Ala145 150 155 160Thr Gln Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala 165 170 175Ala Ile Ala Asp Ile Ile Ser Asp Val Ala Asp Ile Ile Lys Gly Thr 180 185 190Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly Leu Lys 195 200 205Glu Leu Trp Asp Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg 210 215 220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly Val Pro Gly Leu Thr225 230 235 240Gly Ala Thr Ser Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala Ala 245 250 255Gly Leu Ser Ala Ser Ser Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260 265 270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly Gly Gly Ser Gly Phe 275 280 285Gly Gly Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290 295 300Ala Leu Arg Pro Arg Ala Asp Gly Pro Val Gly Ala Ala Ala Glu Gln305 310 315 320Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln Gly Met 325 330 335Gly Gly Pro Val Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser 340 345 350Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu Gly Ala Ala Ala Gly Thr 355 360 365Glu Asp Ala Glu Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln 370 375 380Lys Val Leu Val Arg Asn Val Val385 39011106PRTMycobacterium tuberculosis 11Met Arg His Met Ser Glu Thr Ser Glu Thr Pro Thr Pro Pro Pro His1 5 10 15Gln Thr Pro Lys Val Phe Lys Ala Ala Ala Trp Val Ala Ile Ala Ala 20 25 30Gly Thr Val
Phe Ile Val Ala Val Ile Phe Phe Thr Gly Tyr Ile Leu 35 40 45Gly Lys His Ala Gly His Gly Gly Phe His His Arg Gln His His Gln 50 55 60His Pro Ala Met Met Leu Arg Pro Gly Ser Pro His Gly Gly Pro Ala65 70 75 80Ala Val Arg Pro Gly Pro Gly Pro Gly Gly Pro Gly Gln Val Pro Ser 85 90 95Ser Val Ser Pro Pro Ala Thr Pro Ala Pro 100 10512209PRTMycobacterium tuberculosis 12Met Val Arg Pro Pro Gln Thr Ala Arg Ser Glu Arg Thr Arg Glu Ala1 5 10 15Leu Arg Gln Ala Ala Leu Val Arg Phe Leu Ala Gln Gly Val Glu Ala 20 25 30Thr Ser Ala Glu Gln Ile Ala Glu Asp Ala Gly Val Ser Leu Arg Thr 35 40 45Phe Tyr Arg His Phe Arg Ser Lys His Asp Leu Leu Phe Ala Asp Tyr 50 55 60Asp Ala Gly Leu His Trp Phe Arg Ala Ala Leu Asp Ala Arg Pro Ala65 70 75 80Asp Glu Ser Ile Ile Asp Ser Val Gln Ala Ala Ile Phe Ser Phe Pro 85 90 95Tyr Asp Val Asp Ala Val Thr Lys Ile Ala Ser Leu Arg Arg Gly Glu 100 105 110Leu Glu Pro Ser Arg Ile Val Arg His Met Arg Glu Val Glu Ala Asp 115 120 125Phe Ala Asp Ala Ile Gln Ala Gln Leu Arg Arg Arg Asn Cys Asp Ile 130 135 140Ala Gly Ala Pro Asp Ala Arg Leu His Ile Ala Val Thr Ala Arg Cys145 150 155 160Val Ala Ala Ala Val Phe Gly Ala Met Glu Ala Trp Met Leu Gly Ser 165 170 175Asp Arg Ser Leu Gly Glu Leu Ala Arg Val Cys His Val Ala Leu Glu 180 185 190Ser Leu Arg Val Gly Ile Ser Asp Thr Trp Thr Thr Leu Thr Val Ser 195 200 205Ser13933DNAMycobacterium tuberculosis 13atgacgggac cacaccccga aacagagagc tccggtaacc ggcagatctc ggtggccgag 60ttgctggcca ggcaaggggt caccggcgcc ccggcccgac ggcgccggcg gcgacgcggc 120gatagtgacg ccatcacggt cgccgagctg accggtgaga ttccgatcat tcgtgacgac 180catcaccacg ccggcccgga cgcgcacgcg agccagtctc cggcggctaa cgggcgagtc 240caggttggcg aagctgcccc acagtcgccg gcggaaccag tcgccgagca ggttgccgaa 300gagccaacga gaaccgtgta ctggtcgcaa cccgagccgc gctggcccaa gtcccccccg 360caggaccggc gcgagtccgg gcccgagctt agcgagtacc cgcggccact gcgccacacg 420catagcgaca gagcacccgc ggggccgccg tccggtgccg aacacatgag tccggatccg 480gtcgagcact accccgatct ctgggtggat gtcctggaca ccgaggtggg cgaagcggaa 540gccgagaccg aggtgcgcga agcgcaacct gggcgcggcg agcgccacgc cgcagcggcg 600gcggccggca ccgacgtcga gggtgatggt gcggccgagg cgcgggttgc ccgtcgtgcc 660ctggacgtgg tcccgacgct gtggcgcggc gcgttggtcg tgctgcagtc gatcctggcc 720gttgccttcg gtgccgggtt gttcatcgcc ttcgaccagt tgtggcgctg gaacagcata 780gtggcgctag tgctatcggt gatggtcatc cttggcctag tggtctcggt gcgggcagtc 840cgcaagaccg aagacatcgc cagtacgttg atcgcggttg cggtgggggc gctgattacc 900ctgggaccgc tggccttgtt gcaatcgggc tag 93314546DNAMycobacterium tuberculosis 14gtgagagttt tgttgctggg accgcccggg gcgggcaagg ggacgcaggc ggtgaagctg 60gccgagaagc tcgggatccc gcagatctcc accggcgaac tcttccggcg caacatcgaa 120gagggcacca agctcggcgt ggaagccaaa cgctacttgg atgccggtga cttggtgccg 180tccgacttga ccaatgaact cgtcgacgac cggctgaaca atccggacgc ggccaacgga 240ttcatcttgg atggctatcc acgctcggtc gagcaggcca aggcgcttca cgagatgctc 300gaacgccggg ggaccgacat cgacgcggtg ctggagtttc gtgtgtccga ggaggtgttg 360ttggagcgac tcaaggggcg tggccgcgcc gacgacaccg acgacgtcat cctcaaccgg 420atgaaggtct accgcgacga gaccgcgccg ctgctggagt actaccgcga ccaattgaag 480accgtcgacg ccgtcggcac catggacgag gtgttcgccc gtgcgttgcg ggctctggga 540aagtag 54615153DNAMycobacterium tuberculosis 15atggattttg tgatccagtg gtcgtgctac ctgctggcgt tcctgggggg ctcggctgtt 60gcctgggtag tcgtcactct gtcgatcaag cgcgccagcc gtgatgaggg tgctgcggag 120gcgcccagtg cagccgagac aggcgcacag tga 15316318DNAMycobacterium tuberculosis 16gtgaacgcgc cgttgcgtgg tcaggtctat cgatgcgacc tcggatacgg ggccaaaccg 60tggctcatcg tctccaacaa cgcccgcaac cgtcacaccg ccgacgtggt ggctgtgcgc 120ctgacaacaa cgcggagaac cataccgacc tgggtcgcca tgggccccag cgatccattg 180accggatacg tcaacgcgga caacatcgag accctcggca aagacgagct cggtgactac 240ctcggtgagg tcacgccggc gacgatgaac aaaatcaaca cggcgctcgc gaccgcgctg 300gggctaccgt ggccatga 31817903DNAMycobacterium tuberculosis 17atggatcaac agagtacccg caccgacatc accgtcaacg tcgacggctt ctggatgctt 60caggcgctac tggatatccg ccacgttgcg cctgagttac gttgccggcc gtacgtctcc 120accgattcca atgactggct aaacgagcac ccggggatgg cggtcatgcg cgagcagggc 180attgtcgtca acgacgcggt caacgaacag gtcgctgccc ggatgaaggt gcttgccgca 240cctgatcttg aagtcgtcgc cctgctgtca cgcggcaagt tgctgtacgg ggtcatagac 300gacgagaacc agccgccggg ttcgcgtgac atccctgaca atgagttccg ggtggtgttg 360gcccggcgag gccagcactg ggtgtcggcg gtacgggttg gcaatgacat caccgtcgat 420gacgtgacgg tctcggatag cgcctcgatc gccgcactgg taatggacgg tctggagtcg 480attcaccacg ccgacccagc cgcgatcaac gcggtcaacg tgccaatgga ggagatgcta 540gaggcaacga agtcgtggca ggaatcgggg tttaacgtct tctccggcgg agatctgcgc 600cgaatgggca tcagtgccgc gacggtggcc gcgctggggc aggcgttgtc ggatcccgcg 660gccgaggtcg cagtgtatgc gcgacagtac cgagacgacg ccaagggccc cagcgcctcg 720gtgttgtcgc tgaaagacgg ctccggtgga cgcatcgcgc tgtatcagca ggcgcgaacg 780gcaggttccg gcgaggcgtg gctggctatc tgcccggcta ccccgcagtt ggtgcaagta 840ggagtgaaga ccgttttgga tacactgccc tacggcgagt ggaaaacaca cagcagagta 900tga 90318687DNAMycobacterium tuberculosis 18gtgcgcatca agatcttcat gctggtcacg gctgtcgttt tgctctgttg ttcgggtgtg 60gccacggccg cgcccaagac ctactgcgag gagttgaaag gcaccgatac cggccaggcg 120tgccagattc aaatgtccga cccggcctac aacatcaaca tcagcctgcc cagttactac 180cccgaccaga agtcgctgga aaattacatc gcccagacgc gcgacaagtt cctcagcgcg 240gccacatcgt ccactccacg cgaagccccc tacgaattga atatcacctc ggccacatac 300cagtccgcga taccgccgcg tggtacgcag gccgtggtgc tcaaggtcta ccagaacgcc 360ggcggcacgc acccaacgac cacgtacaag gccttcgatt gggaccaggc ctatcgcaag 420ccaatcacct atgacacgct gtggcaggct gacaccgatc cgctgccagt cgtcttcccc 480attgtgcaag gtgaactgag caagcagacc ggacaacagg tatcgatagc gccgaatgcc 540ggcttggacc cggtgaatta tcagaacttc gcagtcacga acgacggggt gattttcttc 600ttcaacccgg gggagttgct gcccgaagca gccggcccaa cccaggtatt ggtcccacgt 660tccgcgatcg actcgatgct ggcctag 68719324DNAMycobacterium tuberculosis 19gtgtccactt acagatcacc ggatcgcgct tggcaggcgc tggcggacgg cactcgccgg 60gccatcgtgg agcggctggc gcacggcccg ctggccgtcg gcgagttggc ccgcgacctg 120cccgtcagcc gacccgcggt gtcacagcac ctcaaagtgc tcaagaccgc caggctggtg 180tgcgaccgcc ccgcgggaac acgccgcgtc taccagctcg acccgacagg ccttgcggca 240ttgcgcaccg acctcgaccg gttctggaca cgcgccctga ctggctacgc gcagctcatc 300gactccgaag gagacgacac atga 32420816DNAMycobacterium tuberculosis 20atggcgaact tcactgccgc tgacgtcaag cggctgaggg agctgaccgg cgccggcatg 60ctcgcctgta agaacgcgct ggccgaaacc gacggggact tcgacaaggc cgtcgaggcg 120ctgcggatca agggcgctaa ggatgtcggc aagcgtgccg agcgggctac ggccgagggc 180ttggtggcgg ccaaggacgg cgcgctgatc gagctcaact gcgagactga ctttgttgcc 240aagaacgcgg agttccaaac gctggccgac caagtggtcg cggcggcggc agcagctaag 300cccgccgacg tcgacgcgct caagggtgcc agcatcggcg acaagaccgt cgagcaggcg 360atcgccgagc tgtcggccaa gatcggcgag aagctcgagc tgcgtcgtgt ggcgattttc 420gacgggaccg tggaagccta cctgcatcga cgttccgctg acctgccgcc agcggtgggt 480gtactggtcg agtaccgcgg cgacgacgcg gccgccgcgc acgccgttgc gttgcaaatc 540gccgcgctgc gggcgcggta cctgtcccgc gacgacgtgc ctgaagacat cgtggccagc 600gaacgccgca tcgccgagga gacggcaagg gccgagggca agccggagca ggcgctgccc 660aagattgtcg agggccggct gaacggcttc ttcaaggatg cggtgctgct tgagcaggcg 720tcggtgtccg acaataagaa gaccgtcaag gccctgctcg acgtggccgg cgtgacggtg 780acacggttcg tccgcttcga ggtgggccag gcttag 816211347DNAMycobacterium tuberculosis 21ttgaccggcc ccgaacatgg ctccgcctcg accatcgaga tcctgcccgt catcgggctg 60cccgaattcc gtcccggcga cgatctgagc gccgccgtcg ccgcggcggc accgtggcta 120cgcgacggtg acgtcgtggt ggttaccagc aaggtggtgt ccaaatgcga gggccggctg 180gttccggctc ccgaagaccc cgagcaaaga gaccgattgc gccgcaagct gatcgaggat 240gaggcagtgc gcgtgttggc gcgcaaggac cgcacgttga tcaccgagaa tcgactcggg 300ctggttcagg cggccgccgg cgtggacgga tccaacgtcg gccggtccga gttagcgctg 360ctgccggtcg atcctgacgc cagtgccgca accttgcgcg ccgggctgcg cgagcggctc 420ggcgtcaccg tcgccgtggt catcaccgac accatgggac gcgcctggcg caacggccag 480accgatgccg cagtcggcgc tgccggtctg gcggtgctgc gcaactatgc cggtgtccgc 540gacccatacg gcaatgagtt ggtggtcacc gaggtcgcag tcgccgacga gatcgccgcg 600gccgccgact tggtcaaagg caaactgacc gcgacgccgg tggcggtggt gcgtgggttc 660ggcgtgtccg acgacggctc gacagcccgg caactgctgc ggccgggcgc caacgacctg 720ttctggctcg ggaccgccga agcgctcgag ctgggtcgcc agcaagccca actgttgcgc 780aggtccgttc gccggtttag caccgatccg gtgccgggcg acctcgtcga ggctgcggtc 840gccgaggccc tcaccgcgcc agccccacat cacacccggc cgacccgatt cgtgtggctg 900cagacaccgg ccatccgcgc gcggctgcta gatcggatga aagacaagtg gcggtctgat 960ctcaccagtg acggcttgcc cgccgacgcg atagaacgcc gggtggcacg cggccagatc 1020ctctatgacg cacccgaagt cgtcataccg atgctggtgc ccgacggagc acacagctac 1080cccgatgccg cccgcaccga cgccgagcac accatgttca cggtcgccgt cggagcggcc 1140gtacaagcct tgctggtcgc gctggccgtg cgcgggctgg gcagttgctg gatcggctcg 1200acgatctttg ccgctgacct ggtccgcgac gagctggacc tgccagtcga ctgggagccg 1260ttgggcgcca tcgcgatcgg atatgccgac gagccgtccg ggttgcgcga cccggtgcct 1320gccgccgatt tgctgatcct gaagtga 1347221178DNAMycobacterium tuberculosis 22tgagcagagc gttcatcatc gatccaacga tcagtgccat tgacggcttg tacgaccttc 60tggggattgg aatacccaac caagggggta tcctttactc ctcactagag tacttcgaaa 120aagccctgga ggagctggca gcagcgtttc cgggtgatgg ctggttaggt tcggccgcgg 180acaaatacgc cggcaaaaac cgcaaccacg tgaatttttt ccaggaactg gcagacctcg 240atcgtcagct catcagcctg atccacgacc aggccaacgc ggtccagacg acccgcgaca 300tcctggaggg cgccaagaaa ggtctcgagt tcgtgcgccc ggtggctgtg gacctgacct 360acatcccggt cgtcgggcac gccctatcgg ccgccttcca ggcgccgttt tgcgcgggcg 420cgatggccgt agtgggcggc gcgcttgcct acttggtcgt gaaaacgctg atcaacgcga 480ctcaactcct caaattgctt gccaaattgg cggagttggt cgcggccgcc attgcggaca 540tcatttcgga tgtggcggac atcatcaagg gcaccctcgg agaagtgtgg gagttcatca 600caaacgcgct caacggcctg aaagagcttt gggacaagct cacggggtgg gtgaccggac 660tgttctctcg agggtggtcg aacctggagt ccttctttgc gggcgtcccc ggcttgaccg 720gcgcgaccag cggcttgtcg caagtgactg gcttgttcgg tgcggccggt ctgtccgcat 780cgtcgggctt ggctcacgcg gatagcctgg cgagctcagc cagcttgccc gccctggccg 840gcattggggg cgggtccggt tttgggggct tgccgagcct ggctcaggtc catgccgcct 900caactcggca ggcgctacgg ccccgagctg atggcccggt cggcgccgct gccgagcagg 960tcggcgggca gtcgcagctg gtctccgcgc agggttccca aggtatgggc ggacccgtag 1020gcatgggcgg catgcacccc tcttcggggg cgtcgaaagg gacgacgacg aagaagtact 1080cggaaggcgc ggcggcgggc actgaagacg ccgagcgcgc gccagtcgaa gctgacgcgg 1140gcggtgggca aaaggtgctg gtacgaaacg tcgtctaa 117823321DNAMycobacterium tuberculosis 23atgcgacaca tgagtgaaac atctgaaacc ccaacaccac cgcctcacca gacccccaag 60gtgttcaaag ccgcggcgtg ggtagccatt gccgccggaa ccgtgttcat cgtggcggtc 120atcttcttca ccggctatat cctcggcaag catgccggtc acggcggctt ccaccaccgg 180caacaccacc agcacccggc gatgatgctg cgaccgggca gtccgcacgg tgggcccgcg 240gcagtgcgtc ccggcccagg cccgggcggg cccggccagg tgccgtcgtc cgtcagtcct 300cccgcgacac ccgctccgta a 32124630DNAMycobacterium tuberculosis 24atggtccggc ccccgcagac ggcgcgcagc gaacgcactc gcgaggcact gcgccaggcc 60gccctggtgc gatttctggc ccagggcgtc gaggctacct cagccgagca gattgcggag 120gacgccgggg tgtcgctgcg tactttctat cgccacttca gatccaagca cgatttgctg 180tttgccgact acgacgccgg actgcactgg tttcgcgcgg cgctggatgc tagaccggcc 240gacgaatcga tcatcgattc cgtgcaagcg gctatcttct cgtttcctta tgacgttgac 300gcagtgacga agattgcgtc gttgcggcgc ggcgagttgg agccgagccg gatcgtccgc 360catatgcgcg aggtggaagc cgacttcgcc gatgccatcc aggcccaatt gcggcgacgt 420aactgcgata tcgcgggtgc acccgatgcc cgattgcaca tcgccgtcac cgcacgatgc 480gtcgccgcgg cggtgttcgg cgcgatggaa gcctggatgc ttgggagtga ccggtcgctc 540ggggagctgg cgcgggtctg ccacgtcgca ctggagtcgc tgcgcgtcgg catctccgac 600acctggacca ccctcacagt ttcgtcataa 6302535DNAArtificial SequenceSynthetic oligonucleotide 25gaaggagata taccatgcat catcatcatc atcat 352620DNAArtificial SequenceSynthetic oligonucleotide 26tgatgatgag aacccccccc 2027169DNAArtificial SequenceSynthetic oligonucleotide 27cggtcacgct tgggactgcc ataggctggc ccggtgatgc cggccacgat gcgtccggcg 60tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac 120ggtttccctc tagaaataat tttgtttaac tttaagaagg agatatacc 16928238DNAArtificial SequenceSynthetic oligonucleotide 28ggggggggtt ctcatcatca tcatcatcat taataaaagg gcgaattcca gcacactggc 60ggccgttact agtggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg 120ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt 180ttttgctgaa aggaggaact atatccggag cgactcccac ggcacgttgg caagctcg 238
Patent applications by Alan Greener, San Diego, CA US
Patent applications in class Disclosed amino acid sequence derived from bacterium (e.g., Mycoplasma, Anaplasma, etc.)
Patent applications in all subclasses Disclosed amino acid sequence derived from bacterium (e.g., Mycoplasma, Anaplasma, etc.)