Patent application title: TUBERCULOSIS NUCLEIC ACIDS, POLYPEPTIDES AND IMMUNOGENIC COMPOSITIONS
David Roth (San Diego, CA, US)
Huaping He (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.) bacterium or component thereof or substance produced by said bacterium (e.g., legionella, borrelia, anaplasma, shigella, etc.) mycobacterium (e.g., mycobacterium tuberculosis, calmette-guerin bacillus (i.e., bcg), etc.)
Publication date: 2010-05-06
Patent application number: 20100112008
The present invention provides transcriptionally active Mtb
polynucleotides, recombinant Mtb peptides and polypeptides, and
immunogenic Mtb antigens. Immunogenic compositions are also provided that
may be useful as recombinant, subunit and DNA vaccines. In addition the
invention provides diagnostic kits for Mtb.
1. An isolated polynucleotide encoding a Mtb polypeptide that is antigenic
in a mammal, wherein the polynucleotide is selected from the group
consisting of: (a) SEQ ID NOS: 46-64, 110-121; or (b) a fragment thereof,
wherein the fragment encodes an antigenic peptide epitope.
2. The isolated polynucleotide of claim 1, wherein said mammal is a rabbit, human or mouse.
4. The polynucleotide of claim 1, wherein said Mtb polypeptide reacts with polyclonal antibodies directed to Mtb.
5. The polynucleotide of claim 1, wherein said Mtb polypeptide is detected by either ELISA or Western blotting using a polyclonal antibody directed to Mtb.
6. The isolated polynucleotide of claim 1, further comprising: (a) a 5' TAP polynucleotide sequence; and (b) a 3' TAP polynucleotide sequence, wherein said 5' TAP polynucleotide sequence and said 3' TAP polynucleotide sequence are operably coupled to said isolated polynucleotide.
7. The isolated polynucleotide of claim 6, wherein the Mtb polynucleotide sequence is selected from the group consisting of: SEQ ID NOS: 46-64, 110-121.
8. The isolated polynucleotide of claim 6, wherein the 5' TAP polynucleotide sequence comprises a promoter.
9. The isolated polynucleotide of claim 6, wherein the 5' TAP polynucleotide sequence is selected from the group consisting of: SEQ ID NOS: 2, 3, 6, and 84.
10. The isolated polynucleotide of claim 6, wherein the 3' TAP polynucleotide sequence comprises a terminator.
11. The isolated polynucleotide of claim 6, wherein the 3' TAP polynucleotide sequence is selected from the group consisting of: SEQ ID NOS: 4, 5, 7, and 85.
12. A primer pair for amplifying a Mtb polynucleotide selected from the group consisting of: SEQ ID NOS: 8 and 9; 10 and 11; 12 and 13; 14 and 15; 16 and 17; 18 and 19; 20 and 21; 22 and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; 32 and 33; 34 and 35; 36 and 37; 38 and 39; 40 and 41; 42 and 43; 44 and 45; 86 and 87; 88 and 89; 90 and 91; 92 and 93; 94 and 95; 96 and 97; 98 and 99; 100 and 101; 102 and 103; 104 and 105; 106 and, 107; 108 and 109.
13. A method of generating an immune response in a mammalian host against Mtb comprising: administering to said mammalian host an immunogenic composition comprising at least one nucleic acid selected from the group of SEQ ID NO: 46-64, 110-121, fragments thereof or combinations thereof, wherein said nucleic acid encodes and expresses in vivo at least one immunogenic peptide or polypeptide, whereby said immune response against Mtb is generated.
15. The method according to claim 13, wherein said immunogenic composition further comprises and adjuvant.
18. A kit comprising: a) at least one Mtb immunogenic composition selected from a nucleic acid selected from the group consisting of SEQ ID NO: 46-64, 110-121, fragments thereof or combinations thereof.
19. The kit of claim 18, wherein the kit further comprises an expression system.
20. The kit of claim 18 comprising at least two of said immunogenic compositions.
21. The kit of claim 20, comprising at least two of said nucleic acids.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of and claims the benefit of priority to U.S. application Ser. No. 11/291,616 filed on Nov. 30, 2005, entitled TUBERCULOSIS NUCLEIC ACIDS, POLYPEPTIDES AND IMMUNOGENIC COMPOSITIONS, which claims the benefit of priority to U.S. Application Ser. No. 60/632,573 filed on Dec. 1, 2004 and U.S. Application Ser. No. 60/730,951, filed Oct. 26, 2005. The contents of all the aforementioned applications are hereby expressly incorporated by reference, in their entireties.
REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM 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.033C1.txt, created Oct. 27, 2009 which is 101 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
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. But the manufacture and administration of the pathogenic organism carries a risk of infection by the very pathogen the vaccine is designed to treat. Furthermore, recent well-publicized problems with influenza vaccine production highlight 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.
Tuberculosis is a chronic infectious disease that kills approximately 3 million people per year. It has been estimated that two billion people are infected with M. tuberculosis worldwide, including 7.5 million with active cases of tuberculosis. 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.
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 e.g. 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.
In the current age, where treatment of tuberculosis is becoming more challenging and immunosuppressive diseases are more prevalent, new 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 TB in the face of other diseases such as HIV.
SUMMARY OF THE INVENTION
The present invention provides isolated polynucleotides encoding a Mtb polypeptide that is antigenic in any mammal, including SEQ ID NOS: 46-64, 110-121, and fragments thereof, that encode antigenic polypeptides. The mammal can be, for example, a mouse, rabbit, non-human primate, or human. The invention also provides isolated polynucleotides encoding highly immunogenic Mtb antigens including SEQ ID NOS: 46-64, 110-121 and fragments thereof that encode highly immunogenic polypeptides. In some embodiments, highly immunogenic Mtb antigens react with polyclonal antibodies directed to Mtb bacteria (Mtb) from at least two different species. In another embodiment, highly immunogenic Mtb antigens are detected by ELISA, Western blotting, or both using polyclonal antibodies that are directed to Mtb bacteria.
The present invention also provides TAP polynucleotides, e.g. polynucleotides produced by Transcriptionally-Activated PCR (TAP) technology as described in U.S. Pat. No. 6,280,977, which is expressly incorporated herein by reference. Such polynucleotides include a 5' TAP polynucleotide sequence, a Mtb polynucleotide sequence, and a 3' TAP polynucleotide sequence. The Mtb polynucleotide sequence can, for example, comprise one of SEQ ID NOS: 46-64 and 110-121. In some embodiments, the 5' TAP polynucleotide sequence comprises a promoter. In certain embodiments, the 5' TAP polynucleotide sequence is selected from SEQ ID NOS: 2, 3, 6, and 84. In some embodiments the 3' TAP polynucleotide sequence comprises a terminator. In certain embodiments, the 3' TAP polynucleotide sequence is selected from SEQ ID NOS: 4, 5, 7, and 85.
The present invention also provides primer pairs for amplifying an Mtb polynucleotide. These primer pairs include SEQ ID NOS: 8 and 9; 10 and 11; 12 and 13; 14 and 15; 16 and 17; 18 and 19; 20 and 21; 22 and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; 32 and 33; 34 and 35; 36 and 37; 38 and 39; 40 and 41; 42 and 43; 44 and 45; 86 and 87; 88 and 89; 90 and 91; 92 and 93; 94 and 95; 96 and 97; 98 and 99; 100 and 101; 102 and 103; 104 and 105; 106 and 107; 108 and 109.
Isolated antigenic Mtb peptides and polypeptides are encompassed by the invention, including SEQ ID NOS: 65-83, 122-133, and fragments thereof. Isolated Mtb peptides and polypeptides that are highly immunogenic, including SEQ ID NOS: 65-83, 122-133, and fragments thereof, that are highly immunogenic, are also included in the invention. In one embodiment immunogenic peptides and polypeptides react with polyclonal antibodies that are directed to Mtb bacteria (Mtb). In one aspect of this embodiment, the peptides and polypeptides react with polyclonal antibodies that are directed to Mtb bacteria from at least two different species. In another embodiment, highly immunogenic peptides and polypeptides are detected by ELISA, Western blotting or both using polyclonal antibodies that are directed to Mtb bacteria.
The present invention also includes recombinant Mtb peptides and polypeptides, wherein the amino terminus of the peptide or polypeptide comprises an HA tag or a (6×)His tag, and the carboxy terminus of the polypeptide is selected from the group consisting of: SEQ ID NOS: 65-83 and 122-133. Also included are recombinant Mtb peptides and polypeptides, wherein the carboxy terminus of the polypeptide comprises a HA tag or a His tag, and the amino terminus of the polypeptide is selected from the group consisting of: SEQ ID NOS: 65-83 and 122-133. The peptides and polypeptides of the invention may be expressed in an appropriate in vitro transcription and translation system, such as a T7 polymerase system.
Immunogenic compositions for inducing an immunological response in a mammalian host against Mtb are also included in the invention. In one embodiment, the immunogenic compositions comprise nucleic acids that encode and express in vivo in a mammalian host cell at least one immunogenic peptide or polypeptide, which may be any one of SEQ ID NOS: 65-83, 122-133, or immunogenic fragments thereof. The nucleic acids can be, for example, plasmids or TAP fragments. The compositions can induce either a humoral- or cell-mediated immune response. Furthermore, the immunogenic compositions can include additional components, such as adjuvants, as well as other applications such as serodiagnostics.
Immunogenic compositions for inducing an immunological response in a mammalian host against Mtb of the invention can also comprise isolated Mtb peptides and/or polypeptides, such as SEQ ID NOS: 65-83, 122-133 and immunogenic fragments thereof. In one embodiment, the immunogenic peptides and polypeptides are expressed in an in vitro transcription and translation system. The immunogenic peptide and polypeptide compositions can induce either a humoral- or cell-mediated immune response. Furthermore, the immunogenic peptide and polypeptide compositions can include additional components, such as adjuvants, and include other applications such as diagnostics.
Similarly, detection of Mtb, its constituent proteins, and/or its immunologically reactive products (e.g. antibodies) is clinically relevant for the diagnosis of Mtb, and to track the efficacy of therapeutic treatments for Mtb, especially as translated to serodiagnostic tests. The present application therefore provides antigens for detection of Mtb for immune assays, including humoral immune assays. These antigens are applicable to detection of active Mtb in the face of HIV- and other Mycobacterial-coinfections, multi-drug resistant infections by Mtb (MDR1), and rapidly mutating forms of Mtb depending on genetic makeup, geographical location, and immunocompetency status.
As such, the present invention also provides diagnostic compositions, including one or more antibodies directed against the peptide epitopes identified herein. Also, the present invention provides diagnostic kits that include at least one or more of such antibodies.
In addition, the invention provides a method of generating an immune response in a mammalian host against Mtb. The method includes administering to said mammalian host an immunogenic composition comprising at least one nucleic acid selected from the group of SEQ ID NO: 46-64, 110-121, fragments thereof or combinations thereof, wherein said nucleic acid encodes and expresses in vivo at least one immunogenic peptide or polypeptide, whereby said immune response against Mtb is generated.
Also, the invention provides a method of generating an immune response in a mammalian host against Mtb, the method including administering to said mammalian host an immunogenic composition comprising at least one nucleic acid encoding and expressing in vivo at least one immunogenic peptide or polypeptide selected from the group of SEQ ID NO: 65-83, 122-133, fragments thereof or combinations thereof, whereby said immune response against Mtb is generated.
In addition, the invention provides a method of generating an immune response in a mammalian host against Mtb comprising administering to said mammalian host an immunogenic composition comprising at least one immunogenic peptide or polypeptide selected from SEQ ID NO: 65-83, 122-133, fragment thereof or combinations thereof, whereby said immune response against Mtb is generated.
Also, the invention provides kits. In one embodiment the kits include at least one Mtb immunogenic composition selected from a nucleic acid selected from the group consisting of SEQ ID NO: 46-64, 110-121, fragments thereof or combinations thereof, or a peptide selected from the group consisting of SEQ ID NO: 65-83, 122-133, fragments thereof or combinations thereof and an adjuvant. The kit may also include an expression system. In addition the kit may include at least 2, 5, 10, 15, 20 or more of said immunogenic compositions, including nucleic acids and/or peptides, combinations or fragments thereof. In addition, the kit may include controls, e.g. positive and/or negative controls.
BRIEF DESCRIPTION OF THE 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.
FIG. 5 demonstrates that fluorescent proteins (goat IgG antibody) can be more effectively delivered into either NIH-3 T3 cells (A&B) and human dendritic cells (C&D) with a protein delivery reagent (B&D) as opposed to without a protein delivery reagent (A&C).
FIG. 6 shows the results of scanning the Mtb proteome for antigenic targets of humoral immunity by ELISA and Western blotting.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Before proceeding further with a description of the specific embodiments of the present invention, a number of terms will be defined and described in detail.
Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures, techniques and methods described herein are those known in the art to which they pertain. Standard chemical symbols and abbreviations are used interchangeably with the full names represented by such symbols. Thus, for example, the terms "carbon" and "C" are understood to have identical meaning. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients. Standard techniques may be used for recombinant DNA methodology, oligonucleotide synthesis, tissue culture and the like. Reactions and purification techniques may be performed e.g., using kits according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general or more specific references that are cited and discussed throughout the present specification. All references cited herein are incorporated by reference in their entirety and are not admitted to be prior art. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000)), Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)), which are incorporated herein by reference in their entirety for any purpose.
The terms "polynucleotide" and "nucleic acid (molecule)" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-stranded, double-stranded and triple helical molecules. The following are non-limiting embodiments of polynucleotides: a gene, a gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, cosmids, viruses and other vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentyluracil and 2,6-diaminopurine.
The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
Sugar modifications (e.g., 2'-o-methyl, 2-fluoro and the like) and phosphate backbone modifications (e.g., morpholino, PNA', thioates, dithioates and the like) can be incorporated singly, or in combination, into the nucleic acid molecules of the present invention. In one embodiment, for example, a nucleic acid of the invention may comprise a modified sugar and a modified phosphate backbone. In another embodiment, a nucleic acid of the invention may comprise modifications to sugar, base and phosphate backbone.
"Oligonucleotide" refers generally to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded nucleic acid, typically DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or synthesized (e.g., chemically or enzymatically) by methods known in the art. A "primer" refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis.
"Peptide" generally refers to a short chain of amino acids linked by peptide bonds. Typically peptides comprise amino acid chains of about 2-100, more typically about 4-50, and most commonly about 6-20 amino acids. "Polypeptide" generally refers to individual straight or branched chain sequences of amino acids that are typically longer than peptides. "Polypeptides" usually comprise at least about 100 to about 1000 amino acids in length, more typically at least about 150 to about 600 amino acids, and frequently at least about 200 to about 500 amino acids. "Proteins" include single polypeptides as well as complexes of multiple polypeptide chains, which may be the same or different. Multiple chains in a protein may be characterized by secondary, tertiary and quaternary structure as well as the primary amino acid sequence structure; may be held together, for example, by disulfide bonds; and may include post-synthetic modifications such as, without limitation, glycosylation, phosphorylation, truncations or other processing. Antibodies such as IgG proteins, for example, are typically comprised of four polypeptide chains (i.e., two heavy and two light chains) that are held together by disulfide bonds. Furthermore, proteins may include additional components such as associated metals (e.g., iron, copper and sulfur), or other moieties. The definitions of peptides, polypeptides and proteins include, without limitation, biologically active and inactive forms; denatured and native forms; as well as variant, modified, truncated, hybrid, and chimeric forms thereof. The peptides, polypeptides and proteins of the present invention may be derived from any source or by any method, including, but not limited to extraction from naturally occurring tissues or other materials; recombinant production in host organisms such as bacteria, fungi, plant, insect or animal cells; and chemical synthesis using methods that will be well known to the skilled artisan.
"Polyclonal antibodies" or "antisera" are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as rabbits, mice and goats, may be immunized by injection with an antigen or hapten-carrier conjugate optionally supplemented with adjuvants. Polyclonal antibodies may also be derived from the sera of humans or non-human animals exposed to a pathogen or vaccinated against a pathogen using a commercially available or experimental vaccine. An antiserum against TB (Mtb), for example, may be obtained from a human patient vaccinated with a TBvaccine, or from an animal, such as a mouse, rabbit, goat or sheep immunized with Mtb bacteria or a Mtb preparation.
"Monoclonal antibodies," which are abbreviated MAb, are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110, the human B-cell hybridoma technique (Kosbor, et al., Immunology Today, 4:72 (1983); Cote, et al., Proc. Natl. Acad. Sci. USA, 80:2026-30 (1983), and the EBV-hybridoma technique (Cole, et al., in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the MAb of this invention may be cultivated in vitro or in vivo. Production of high titers of MAbs in vivo makes this a presently preferred method of production
In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Takeda, et al., Nature, 314:452-54 (1985)) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different sources, such as those having a variable region derived from a murine MAb and a human immunoglobulin constant region. Humanized antibodies can also be generated in which certain parts (e.g., framework regions) of a non-human antibody are altered to make the antibody more like a human antibody, while retaining antigen binding features of the parent molecule.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-26 (1988); Huston, et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988); and Ward, et al., Nature, 334:544-46 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are typically formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the Fab fragments that can be produced by papain digestion of the antibody molecule, the F(ab')2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab' fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse, et al., Science, 246:1275-81 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
The term "hapten" as used herein, refers to a small proteinaceous or non-protein antigenic determinant that is capable of being recognized by an antibody. Typically, haptens do not elicit antibody formation in an animal unless part of a larger species. For example, small peptide haptens are frequently coupled to a carrier protein such as keyhole limpet hemocyanin in order to generate an anti-hapten antibody response. "Antigens" are macromolecules capable of generating an antibody response in an animal and being recognized by the resulting antibody. Both antigens and haptens comprise at least one antigenic determinant or "epitope," which is the region of the antigen or hapten that binds to the antibody. Typically, the epitope on a hapten is the entire molecule.
By the terms "specifically binding" and "specific binding" as used herein is meant that an antibody or other molecule, binds to a target such as an antigen, with greater affinity than it binds to other molecules under the specified conditions of the present invention. 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 binding" 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.
"Immunogenic compositions" of the present invention 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.
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. 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.
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.
By "serodiagnostic test" or grammatical equivalents herein is meant diagnostic tests to detect Mtb by serum of infected organisms, animals or patients.
By "diagnostic test" or grammatical equivalents herein is meant an assay or test to detect Mtb by any scientific technique from infected organisms, animals or patients.
The present invention generally relates to Mtb polypeptide libraries, methods of determining the immunogenic effect of Mtb polypeptides, and methods of developing vaccines against Mtb, as well as immunogenic and pharmaceutical compositions. The invention also provides immunogenic Mtb polypeptides and mixtures of polypeptides, polynucleotides encoding immunogenic Mtb polypeptides and immunogenic compositions comprising Mtb polypeptides or polynucleotides.
According to a method of the present invention, a library or array of Mtb polypeptides, oligonucleotides, or polynucleotides is generated. The immunogenicity of individual polypeptides in the library or array is determined by immunological screening where suitable, highly immunogenic Mtb polypeptides are selected for vaccine development. Conveniently, individual polypeptides in the library may be arranged in an array to facilitate screening in a rapid and high throughput manner.
The term "array" includes any arrangement wherein a plurality of different molecules, compounds or other species are contained, held, presented, positioned, situated, or supported. Arrays can be arranged on microtiter plates, such as 48-well, 96-well, 144-well, 192-well, 240-well, 288-well, 336-well, 384-well, 432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well, 1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plates, tubes, slides, chips, flasks, or any other suitable laboratory apparatus. In one embodiment, molecules arranged in an array are peptides, polypeptides or proteins. In another embodiment, the molecules are oligonucleotides or polynucleotides. In one aspect of the invention, polypeptides or polynucleotides in solution are arranged in 96 well plate arrays. In another embodiment, polypeptides or polynucleotides are immobilized on a solid support in an array format. Furthermore, an array can be sub-divided into a plurality of sub-arrays, as for example, where multiple 96-well plates (each an individual sub-array) are required to hold all of the samples of a single, large array.
The term "library" is likewise to be construed broadly, and includes any non-naturally occurring collection of molecules, whether arranged or not. A library therefore encompasses an array but the two terms are not necessarily synonymous.
Libraries of Mtb polypeptides may be prepared by any method known in the art. Conveniently, GTS' patented Transcriptionally Active PCR ("TAP") products can be used to amplify DNA in preparation for producing Mtb polypeptide libraries. 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 of the invention, 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: 84) 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: 85) 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.
Bioinformatics Analysis of Mtb Polynucleotides
In one embodiment of the invention, a bioinformatics approach is used to identify, prioritize and select Mtb genes, coding sequences, ORFs and other sequences of interest for TAP amplification and to design custom 5' and 3' oligonucleotide primers. According to this approach, a database of Mtb genomic information is compiled from available nucleic acid and amino acid sequence information, including the polynucleotide, gene, locus, polypeptide, and protein names, locations and sizes. In certain embodiments, the location of known coding sequences is included in the database. The sequence information may also be analyzed for unidentified ORFs and putative coding sequences. Any method can be used to identify ORFs and coding sequences including free or commercially available sequence analysis software. For example, the GLIMMER program may be used to predict putative coding regions or genes in prokaryotic nucleotide sequences. See e.g., Salzberg et al., Nucleic Acids Res. 26: 544-548 (1998); Delcher, et al., Nucleic Acids Res. 27:4636-4641 (1999).
In certain aspects of the invention, the genome database includes the entire genomic DNA sequence of Mtb. In one embodiment, the sequence information is obtained from information that is in the public domain. In other embodiments, some or all of the sequence information can be obtained by nucleotide and/or amino acid sequencing.
As previously described in U.S. Ser. No. 10/159,428, which is hereby incorporated by reference in its entirety, the methods of the present invention, particularly TAP technology, enable the skilled artisan to prepare a library representing all or substantially all of the polypeptides expressed in an organism or cell type. In certain embodiments of the present invention, however, it may be preferable to prepare a library of polypeptides with selected properties. Thus, one aspect of the present invention utilizes a set of ranking criteria to identify polypeptides predicted to have properties desirable e.g., for vaccine development. Polypeptide ranking criteria, which may be identified using bioinformatics tools, include but are not limited to, the presence of membrane domains, ORF size, secreted proteins signatures, signal sequences, hydrophobicity, B-cell and T-cell epitopes, homology to human proteins, protein and gene expression levels. The ranking criteria may be assigned a numerical score based on relative importance. Coding regions or putative coding regions identified in the database of Mtb sequences are then scored using the numerical ranking criteria and the sum of the scores for each sequence is used to establish a rank order. According to this aspect of the invention, primers are designed to amplify Mtbpolynucleotides in rank order. A library may be constructed, for example, from the top 5%, 10%, 20%, 30%, 40% or 50% by rank of Mtb polynucleotides.
Amplification of Mtb Polynucleotides
Using the custom 5' and 3' oligonucleotide primers, TAP primary fragment may be amplified by methods well known in the art. The term "TAP primary fragment" refers to an amplified Mtb polynucleotide, and in one embodiment relates to a polynucleotide sequence that has been amplified but is not transcriptionally active. Generation of TAP primary fragments involves performing PCR, which generates a polynucleotide fragment that contains the Mtb polynucleotide sequence with 5'- and 3'-TAP universal end sequences and may contain other sequences incorporated into the custom 5' and 3' oligonucleotide primers. The 5'- and 3'-TAP universal end sequences are particularly useful for incorporating one or more nucleotide sequences into TAP primary fragment that confer transcriptional activity. In one embodiment, these sequences can include TAP Express® promoter and terminator fragments (e.g., SEQ ID NOS: 2-7). The skilled artisan will be familiar with methods for amplifying polynucleotides, (e.g. by using PCR) and can adjust the above methods in order to optimize the amplification reaction.
An additional step in the generation of TAP fragments involves incorporating at least one polynucleotide sequence that confers transcriptional activity into the TAP primary fragment. Typically, at least one polynucleotide sequence is incorporated by performing a second PCR reaction. Examples of polynucleotide sequences that confer transcriptional activity are promoter sequences (e.g., prokaryotic Pribnow boxes and eukaryotic TATA box sequences) binding sites for transcription factors, and enhancers. In one embodiment, one promoter and one terminator sequence are added to the TAP fragment. These promoter and terminator sequences can be obtained in numerous ways. For example, one can use restriction enzyme digestion of commercially available plasmids and cDNA molecules, or one can synthesize these sequences with an automated DNA synthesizer by methods well known in the art.
The end product of the second PCR reaction is referred to as a "TAP expression fragment," which is a transcriptionally active polynucleotide, and which is generally a transcriptionally active coding sequence. In certain embodiments, the TAP expression fragments are used directly for in vivo or in vitro (e.g. cell-free) expression. In other embodiments, TAP expression fragments are transfected into cultured cells or injected into animals.
Generating TAP fragments is a rapid and efficient way of making a large number of polynucleotide sequences transcriptionally active. Accordingly, a plurality of different genes from Mtb can be made transcriptionally active using TAP technology. Thus, a library representing all, substantially all, or a selected subset of the coding sequences in the Mtb genome can be constructed using TAP technology.
TAP Tags and Linker Molecules
As described above, TAP technology provides powerful methods for amplifying and expressing Mtb polynucleotides. Coding sequences can be rendered transcriptionally active by the PCR-mediated addition of promoter sequences, enhancers, terminators and other regulatory sequences.
In addition, Mtb polynucleotides can be amplified with additional coding or non-coding sequences that can facilitate rapid screening, characterization, purification and study of the polypeptides that they encode. These additional sequences include, for example, reporter genes, affinity tags, antibody tags, PNA binding sites, secretory signals, and the like.
According to the present invention, Mtb polynucleotides can be synthesized with an epitope tag. An "epitope tag" is a short stretch of polynucleotide sequence encoding an epitope. In one embodiment, this epitope is preferably recognized by a well-characterized antibody. By incorporating an epitope tag into TAP fragments, the Mtb polynucleotide of interested can be fused in-frame to an epitope-encoding sequence. Expression of an epitope-tagged TAP fragment produces a fusion protein comprising a tagged Mtb polypeptide. Suitable epitope tags will be well known to those skilled in the art, including the hemagglutinin (HA), the 6×His epitope tag, and the Flag epitope tag. The HA epitope tag is well characterized and highly immunoreactive. Upon transfection of an HA-tagged TAP fragment into cells, the resulting HA-tagged polypeptides can be identified with commercially available anti-HA antibodies. Epitope tagging of TAP fragments is useful for rapidly and conveniently detecting expression of TAP fragments. Epitope tagging of TAP fragments can also help determine the intracellular distribution of Mtb polypeptides and help characterize and purify the Mtb polypeptide. Furthermore, epitope-tagged expression products can be quickly captured and/or purified using antibodies specific for the specific epitope. Antibodies directed against the HA epitope can used in the full range of immunological techniques for detection and analysis of tagged polypeptides including but not limited to Western blotting, ELISAs, radioimmune assays, immunoprecipitation, immunocytochemistry and imnuunofluorescence, fluorescence assisted cell sorting (FACS) and immunoaffinity purification of the desired fusion polypeptides.
The present invention also provides Mtb polypeptides fused to affinity tags. For example, a polynucleotide sequence encoding a histidine tag can be incorporated into the TAP fragment to enable the expressed gene product to be conveniently purified. These His tags consist of six consecutive histidine residues (6×His) and are a powerful tool for recombinant polypeptide purification. The 6×His tag interacts with metals, such as nickel. Thus, polypeptides fused to a 6×His tag can be purified by metal affinity chromatography, for example, using a nickel nitrilotriacetic (Ni-NTA) resin. The 6×His tag is much smaller than most other affinity tags and is uncharged at physiological pH. It rarely alters or contributes to polypeptide immunogenicity, rarely interferes with polypeptide structure or function, does not interfere with secretion, does not require removal by protease cleavage, and is compatible with denaturing buffer systems. Accordingly, this tag is a powerful adjunct to expression and purification of recombinant proteins.
In one aspect of this embodiment, TAP primers can be designed to include the nucleotide sequence encoding the 6×His epitope tag: to add the 6×His epitope to the 5' end of a Mtb polynucleotide, a sequence encoding histidine residues can be included along with the promoter-containing primer; to add the 6×His epitope to the 3' end, the His sequence can be included in the terminator-containing primer.
Commercially available nickel affinity resins can be used to purify 6×His tagged polypeptides. For example, the well-established QIAexpress Protein Expression and Purification Systems are based on the remarkable selectivity and affinity of patented nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices for polypeptides tagged with 6 consecutive histidine residues (6×His tag) available from QIAGEN (Seattle, Wash.). The QLAexpress System is based on the remarkable selectivity of Ni-NTA (nickel-nitrilotriacetic acid) for polypeptides with an affinity tag of six consecutive histidine residues--the 6×His tag. This technology allows purification, detection, and assay of almost any 6×His-tagged polypeptide from any expression system. Polypeptides with a 6×His tag can be purified through nickel nitrilotriacetic (Ni-NTA) resin
The HA and the 6×His epitope embodiments are not to be construed as limiting, and are provided for illustrative purposes only. Those skilled in the art will appreciate that any type of tag can be attached to the expressed products such as for example, a 7×, 8×, 9×, or 10×histidine tag, GST tag, fluorescent protein tag, and the like.
In addition to providing a convenient means for detection and purification of Mtb polypeptides, various tags can provide a "linker" through which the polypeptides of the invention can be immobilized on a solid support. The term "linker molecule," as used herein, encompasses any molecule that is capable of immobilizing the polypeptides to a solid support.
TAP Fragment with Secretory Signal
For many gene therapy and DNA vaccine applications it may be beneficial for the gene product to be secreted from the transfected cells. Thus, one embodiment of the invention provides a version of the TAP system designed to express Mtb polypeptides containing a fused a secretory signal. A commonly used signal peptide is the first 23 amino acids from human tissue plasminogen activator (tPA) with the coding sequence as follows: ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GTG CTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC. (SEQ ID NO: 1) This sequence can be built into the TAP promoter fragment to create a new TAP fragment in a fashion similar to the construction of the tagged polypeptides described above.
Incorporating TAP Fragments into a Plasmid Vector
Once the function or immunogenicity of an Mtb polypeptide is identified, it may be of desirable to clone the TAP fragment into a plasmid or other vector to facilitate further gene characterization and manipulation. Standard cloning techniques can involve the use of restriction enzymes to digest the plasmid and the gene fragment to be inserted. Annealing and ligation of the compatible ends can lead to insertion of the gene into the vector. An alternative method of restriction ends-directed cloning is to prepare a linearized plasmid with T overhangs on the 3' ends of the double-stranded DNA to accommodate DNA fragments amplified with the aid of specific polymerases through PCR. This method is sometimes called "T/A cloning". Other methods of cloning TAP fragments will be well known to those of skill in the art.
In certain embodiments, the TAP Cloning systems, methods, and kits can further simplify the cloning process by taking advantage of the universal 5' and 3' sequences that are present on the TAP Express fragment after the first or second PCR step. These regions overlap with the end sequences of our linearized TAP Express Cloning Vector. When the TAP fragment and the linearized plasmid are mixed together and directly electroporated into TAP Express Electro-Comp cells, endogenous bacterial recombinase activity recombines the two DNA fragments resulting in a plasmid with the inserted TAP Express fragment. This process can replace conventional cloning with two simple PCR steps. In some embodiments it does not require cutting, pasting and ligating DNA fragments. In addition, this process can be highly suited for fast and convenient cloning of TAP PCR fragments without having to resort to restriction enzymes, DNA ligase, Topo-isomerase or other DNA modifying enzymes. "TAP" systems, vectors and cells are readily available from Gene Therapy Systems, Inc., San Diego, Calif.
GeneGrip PNA compatible TAP system can also be used to couple polypeptides onto DNA through PNA-Dependent Gene Chemistry, thereby avoiding many of the limitations of previously described methodologies. GeneGrip is available through Gene Therapy Systems, Inc., San Diego, Calif. This approach takes advantage of the property of peptide nucleic acids (PNA) to hybridize with duplex DNA in a sequence specific and very high affinity manner. PNA binding sites can be used for attaching a series of peptides onto DNA in order to target the transfected plasmid and improve transgene expression, for example. This can facilitate a rational approach to improve the efficiency and efficacy of gene delivery by adding elements intended to increase nuclear uptake, facilitate endosomal escape, or target gene delivery to the cell surface or to intracellular receptors.
Incorporating a GeneGrip site into TAP enables peptide nucleic acids (PNAs) to be hybridized to the TAP gene product. Ligands can then be attached to the PNA in order to improve the bioavailability and DNA vaccine potency of the gene.
System for Performing TAP Method
In another embodiment of the invention, a system can be used to perform every step involved in generating TAP fragments from a Tb, and in particular the Mtb genome. Additionally, each individual step is capable of being controlled by a system. For example, a system can design customized PCR primers, obtain said primers, perform PCR reactions utilizing TAP technology, attach promoters and terminators, and attach sequences that encode linker molecules to the primary or expression fragment. The system can be either automated or non-automated. In one embodiment of the invention, the system comprises a computer program linked to robotic technologies for rapid and high throughput gene amplification of the genome.
Expression of the TAP Fragment
TAP fragments can be used directly as templates in various expression systems in order to obtain the corresponding polypeptide for each coding sequence in the Mtb genome. The invention provides simple, efficient methods for generating TAP fragments from Mtb that can be readily transfected into animal cells or tissues by any nucleic acid transfection technique. The methods of the invention can avoid the need for subcloning into expression vectors and for purification of plasmid DNA from bacteria. As skilled artisans can appreciate, TAP fragments can be rapidly expressed using in vivo or in vitro (e.g. cell-free) expression systems. For example, the amplified TAP fragments can be directly transfected into a eukaryotic or prokaryotic cell for expression. Examples of eukaryotic cells that can be used for expression include mammalian, insect (e.g. Baculovirus expression systems), yeast (e.g. Picchia pastoris), and the like. An example of a prokaryotic cell expression system includes E. coli.
Alternatively, expression can be accomplished in cell-free systems, for example, a T7 transcription and translation system. Cell-free translation systems can include extracts from rabbit reticulocytes, wheat germ and Escherichia coli. These systems can be prepared as crude extracts containing the macromolecular components (30S, 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. To promote efficient translation, each extract can be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg.2+, K.sup.+, etc.)
The use of TAP technology allows skilled artisans to rapidly express polypeptides from a plurality of polynucleotides. After a particular Mtb polynucleotide of interest is rendered transcriptionally active, other Mtb polynucleotides can also be made to be transcriptionally active according to the methods of the invention. Accordingly, in one embodiment of the invention, a plurality of polynucleotides from Mtb are amplified and expressed in order to generate a library or array of Mtb polypeptides.
Other embodiments of the invention relate to expressing the product of a Mtb polynucleotide that encodes an epitope tag, affinity tag or other tags, and which may function as linkers. A polynucleotide sequence encoding a linker molecule can be incorporated into a TAP primary fragment or a TAP expression fragment. Accordingly, the linker molecule can be expressed as a fusion to the Mtb polypeptide.
The generation of Mtb polypeptide libraries according to the methods of the invention allows skilled artisans to easily use them in subsequent research and study. For example, it is possible to organize the expressed Mtb polypeptides into an array for further analysis. The expressed polypeptide arrays can be screened in order to identify, for example, new vaccine and drug targets against microbial, neoplastic disease and the like. The expressed polypeptides can be used to screen antibody libraries, to develop unique research reagents, for functional proteomic studies, and the like. These steps can be rapidly accomplished at rates far exceeding traditional methods.
In addition to amplifying Mtb polynucleotides of interest using TAP technology, the present invention also encompasses amplifying Mtb polynucleotides using "adapter technology". In some embodiments adapter technology is performed using a one-step PCR reaction. The term "adapter technology" as used herein relates to methods of cloning a desired polynucleotide into a vector by flanking a desired nucleic acid sequence, a Mtb TAP fragment for example, with first and second adapter sequences. The resulting fragment can be contacted with the vector having sequences homologous to the first and second adapter sequences under conditions such that the nucleic acid fragment is incorporated into the vector by homologous recombination in vivo in a host cell. Accordingly, adapter technology allows for fast and enzyme-less cloning of nucleic acid fragments into vectors and can also be used for forced cloning selection for successful transformation. Adapter technology is described in more detail in U.S. patent application Ser. No. 09/836,436, entitled "Fast and Enzymeless Cloning of Nucleic Acid Fragments", U.S. patent application Ser. No. 10/125,789, entitled "Rapid and Enzymeless Cloning of Nucleic Acid Fragments", and PCT Application No. PCTUS 02/12334, all of which are hereby incorporated by reference in their entirety
The nucleic acid fragment can be incorporated into any vector using adaptor technology. In certain embodiments, the vector that the fragment is incorporated into can be, for example, a plasmid, a cosmid, a bacterial artificial chromosome (BAC), and the like. The plasmid can be CoE1, PR100, R2, pACYC, and the like. The vector can also include a functional selection marker. The functional selection marker can be, for example, a resistance gene for kanamycin, ampicillin, blasticidin, carbonicillin, tetracycline, chloramphenicol, and the like. The vector further can include a dysfunctional selection marker that lacks a critical element, and wherein the critical element is supplied by said nucleic acid fragment upon successful homologous recombination. The dysfunctional selection marker can be, for example, kanamycin resistance gene, ampicillin resistance gene, blasticidin resistance gene, carbonicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, and the like. Further, the dysfunctional selection marker can be, for example, a reporter gene, such as the lacZ gene, and the like.
The vector can include a negative selection element detrimental to host cell growth. The negative selection element can be disabled by said nucleic acid fragment upon successful homologous recombination. The negative selection element can be inducible. The negative selection element can be, for example, a mouse GATA-1 gene. The vector can include a dysfunctional selection marker and a negative selection element.
The host cell used in adapter technology can be a bacterium. The bacterium can be capable of in vivo recombination. Examples of bacterium include JC8679, TB1, DHx, DH5, HB101, JM101, JM109, LE392, and the like. The plasmid can be maintained in the host cell under the selection condition selecting for the functional selection marker.
The first and second adapters can be any length sufficient to bind to the homologous sequences of the vector such that the desired nucleic acid sequence is incorporated into the vector. The first and second adapter sequences can be, for example, at least 11 bp, 12 bp, 13, bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 40 bp, 50 bp, 60 by and the like. Furthermore, the first and second adapter sequences can be greater than 60 bp.
The first and second adapter sequences further can include a functional element. The functional element can include a promoter, a terminator, a nucleic acid fragment encoding a selection marker gene, a nucleic acid encoding a linker molecule, a nucleic acid fragment encoding a known protein, a fusion tag, a nucleic acid fragment encoding a portion of a selection marker gene, a nucleic acid fragment encoding a growth promoting protein, a nucleic acid fragment encoding a transcription factor, a nucleic acid fragment encoding an autofluorescent protein (e.g. GFP), and the like.
When the common sequences on both the 5' and 3' ends of the nucleic acid fragment are complimentary with terminal sequences in a linearized empty vector, and the fragment and linearized vector are introduced, by electroporation, for example, together into a host cell, they recombine resulting in a new expression vector with the fragment directionally inserted. In alternative embodiments the host cell can include the linearized empty vector so that only the nucleic acid fragment is introduced into the host cell. It should be noted that in alternative embodiments of the present invention the vector can be circularized, and as used herein a vector can be either linearized or circular. The host cell is converted into an expression vector through homologous recombination. In principle this approach can be applied generally as an alternative to conventional cloning methods.
A nucleic acid fragment having first and second adapter sequences can be generated by methods well known to those of skill in the art. For example, a gene of interest with known 5' and 3' sequences undergoes PCR along with overlapping 5' and 3' priming oligonucleotides. The priming oligonucleotides can be obtained by methods known in the art, including manufacture by commercial suppliers. A primary fragment with adapter sequences can be generated. The adapter sequences flanking the gene of interest can be homologous to sequences on a vector or to sequences from other 5' or 3' fragments to be used in a subsequent PCR
In some embodiments of the invention, a particular polynucleotide of interest from Mtb can be amplified with an adapter sequence on both the 3' and 5' ends. In other embodiments adapters can be attached to a plurality of polynucleotides, for example every coding region in the Mtb genome. In certain embodiments adapters can make the desired coding regions transcriptionally active. Once incorporated into the desired vector, the Mtb coding region can be rapidly replicated and expressed, such that a plurality of Mtb's genes, for example every gene, is expressed.
Pluralities of expression products can be stored in libraries or arrays and can be assayed for their immunogenic properties as will be discussed below. While most embodiments relating to the assay methodologies are discussed in terms of TAP technology, all of the following assays can be used on adapter technology expression products as well. Once the appropriate assays are conducted on the adapter technology expression products, methods of developing vaccines can be utilized. While most of the embodiments relating to developing vaccines, discussed below, pertain to TAP technology, all of the vaccine embodiments can also be used with polypeptide libraries and arrays resulting from adapter technology.
Identifying Immunogenic Polypeptides
Libraries and arrays of polypeptides, prepared through TAP or adapter technology with subsequent expression can be useful in the development of polypeptide or nucleic acid subunit vaccines. DNA vaccines are effective vaccines that are inexpensive to manufacture, easy and safe to deliver, and can be widely distributed. It has been found that plasmid DNA, when injected into mice without being associated with any adjuvant, can generate antibody and CTL responses to viral antigens encoded by the plasmid DNA, and elicit protective immunity against viral infection (Ulmer at al., Science, 259:1745, 1993). Starting from this, there have been reported many research results regarding the induction of humoral and cellular immune responses resulting from the introduction of DNA vaccines containing various viral genes in animal models (Chow et al., J. Virol., 71:169, 1997; McClements et al., Proc. Natl. Acad. Sci. USA, 93:11414, 1996; Xiang et al., Virology, 199:132, 1994; Wang et al., Virology, 211:102, 1995; Lee et al., Vaccine, 17:473, 1999; Lee et al., J. Virol., 72:8430, 1998). As well, DNA delivery by electroporation techniques has been well-described (Heller et al., Expert Opin. Drug Deliv. 2(2): 1-14, 2005.
One of the most difficult tasks in developing a DNA vaccine (or any recombinant subunit vaccine) to a pathogen such as Mtb, is the identification of antigens that can stimulate the most effective immune response against the pathogen, particularly when the genome of the organism is large.
A comprehensive means to accomplish this task, which is embodied by the present invention, is to obtain a plurality of polypeptides from the particular pathogen in the mode of a library or array. These polypeptides can be tested to determine their capability to evoke a humoral and/or a cell-mediated immune response. Polypeptides that evoke immunogenic responses can be tested individually or with other antigens for effectiveness as subunit vaccines. In addition, nucleic acids that encode identified antigenic polypeptides can be used alone or with other nucleic acids that encode antigens to develop a recombinant vaccine, such as a DNA vaccine, for the particular pathogen.
One embodiment of the invention, incorporates a Rapid High-Throughput Vaccine Antigen Scanning approach, using TAP Express, that is able to systematically screen and identify all, substantially all, or a subset of the antigens in Mtb that give rise to a humoral and cell-mediated immune response. The identification of the Mtb antigens allows for the development of a highly specific subunit vaccine
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
In certain methods of developing a Mtb vaccine, a plurality of Mtb polynucleotides can be made transcriptionally active. In one embodiment, all of the open reading frames from Mtb genome can be made transcriptionally active using TAP technology. The present invention thus provides Mtb polynucleotides (SEQ ID NOS: 46-64, 110-121) that have been made transcriptionally active.
The resulting Mtb TAP fragments of the present invention can be purified and expressed in vitro or in vivo according to any method known in the art. The expression products, which encompass SEQ ID NOS: 65-83, 122-133, can be assayed by various methods to determine their ability to evoke a humoral and/or a cell-mediated immunogenic response. Polypeptides that are identified as capable of evoking an immune response can be used as candidates to develop polynucleotide or polypeptide subunit vaccines. The complete method will be described in more detail below.
According to one embodiment of the present invention, TAP fragments from Mtb are used to generate a DNA array, and then, if desired, a protein array. In certain embodiments, primers are designed for every gene in the Mtb genome. In another embodiment, designing the primers allows a skilled artisan to make any given Mtb polynucleotide transcriptionally active using TAP technology. In yet another embodiment, coding regions, ORFs and other polynucleotide sequences of interest are ranked according to and the top Mtb polynucleotides are made transcriptionally active using TAP technology.
As mentioned above, the custom PCR primers can be designed by using an automated system, such as a computerized robotics system. For example, in order to design custom primers for use in the TAP process, a robotic workstation can be interfaced with a dual Pentium III CPU (1.4 GHz) computer running the Linux operating system. In addition, a customized MySQL database can manage the input sequence data from GenBank and from other sources. This database can track all the operations, samples and analytical data generated by the robot. In another embodiment, PCR primers, PCR products and polypeptides can be tracked by the database. For example, PCR primers, PCR products and polypeptides can be tracked by using bar coded 96-well plates. While the embodiments below discuss using 96-well plates in certain embodiments, those skilled in the art can appreciate that any sized well plate can be used. For example, the well plates can consist of about 48, about 96, about 144, about 192, about 240, about 288, about 336, about 384, about 432, about 480, about 576, about 672, about 768, about 864, about 960, about 1056, about 1152, about 1248, about 1344, about 1440, about 1536 or more wells. In addition to well plates, the PCR products and polypeptides can be tracked using any suitable receptacles, for example test tubes.
Custom oligonucleotide pairs of the present invention (SEQ ID NOS: 8 and 9; 10 and 11; 12 and 13; 14 and 15; 16 and 17; 18 and 19; 20 and 21; 22 and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; 32 and 33; 34 and 35; 36 and 37; 38 and 39; 40 and 41; 42 and 43; 44 and 45; 86 and 87; 88 and 89; 90 and 91; 92 and 93; 94 and 95; 96 and 97; 98 and 99; 100 and 101; 102 and 103; 104 and 105; 106 and 107; 108 and 109), which are needed for the TAP PCR reactions, can be synthesized or obtained in order to perform the TAP technology. In certain embodiments, the Mtb genome sequence data and primer design software (e.g., Primer 3) can be used by the system to generate custom primer pairs for all, substantially all, or a subset of the genes in the Mtb genome. The primers can be organized into arrays of about 48, about 96, about 144, about 192, about 240, about 288, about 336, about 384, about 432, about 480, about 576, about 672, about 768, about 864, about 960, about 1056, about 1152, about 1248, about 1344, about 1440, or about 1536 5' primers and 3' primers according to polynucleotide size and GC content, such PCR reaction conditions can be optimized on a plate by plate basis. The present invention further contemplates that sequences for each of the custom Mtb primer pairs can be sent to an oligonucleotide synthesis provider (e.g., MWG Biotech, Inc., High Point, N.C.) where they can be synthesized. Synthesized primers can be organized and dispensed into bar-coded plates at a desired concentration, such as 100 pmole/μl, frozen and shipped to the practitioner. In one embodiment, 600 Mtb-specific PCR primers, which are capable of amplifying 300 Mtb coding sequences are designed, generated, ordered, and organized
After obtaining or generating the custom Mtb PCR primers, the Mtb polynucleotides of interest can be amplified. In one embodiment, the primers can be organized into arrays of 96 5' primers and 96 3' primers according to polynucleotide size, and placed onto a robotic workstation. The robot can be programmed to generate a plate of about 48, about 96, about 144, about 192, about 240, about 288, about 336, about 384, about 432, about 480, about 576, about 672, about 768, about 864, about 960, about 1056, about 1152, about 1248, about 1344, about 1440, about 1536 PCR reactions by mixing the appropriate 5' and 3' primers with Taq polymerase and Mtb genomic DNA. In addition to Taq, any thermally stable polymerase can be used in the PCR reactions. For example, Vent, Pfu, Tfl, Tth, and Tgo polymerases can be used. The robotic workstation can transfer the PCR reaction plate containing the mixed reagents to a PCR machine for amplification. In one embodiment, the robotic workstation can use a robotic arm to transfer the PCR reaction plate to the PCR machine.
The first TAP PCR procedure can be run for any number of cycles. In one embodiment, the PCR machine is run for about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more cycles. The first TAP PCR reactions can be transferred robotically to a Millipore Montage 96-well cleanup kit, for example, when desired. Any method, kit or system can, however, be used to purify the PCR products from these reactions. According to one embodiment, a vacuum station of the robotic platform can carry out the purification step. In some embodiments, an aliquot of the resulting product can be transferred robotically to an analysis plate containing the Pico-Green fluorescent probe (Molecular Probes, Eugene, Oreg.) that reacts only with the dsDNA products. Depending on the number of wells, the plate can be transferred to an about 48, about 96, about 144, about 192, about 240, about 288, about 336, about 384, about 432, about 480, about 576, about 672, about 768, about 864, about 960, about 1056, about 1152, about 1248, about 1344, about 1440, about 1536 or more well fluorescent plate reader. The fluorescent signal can be compared to a standard curve to determine the amount of double stranded PCR product generated in this first PCR step. Persons with skill in the art can adjust the above methods in order to optimize their particular PCR reaction, should the need arise.
In addition to the first TAP PCR procedure, a second TAP PCR reaction can be performed to add at least one sequence that confers transcriptional activity to the primary TAP primary fragment. In one embodiment, a robot can be programmed to transfer an aliquot of each TAP primary fragment from the first TAP PCR reaction into a PCR reaction containing a promoter- and a terminator-containing primers. In a particular embodiment, the promoter can be a T7-his tag promoter sequence and the terminator can be a T7-His tag terminator sequence. Those with skill in the art can appreciate that any promoter or terminator sequence can be added to the primary transcript. In addition, any polynucleotide sequence that encodes a tag or linker allowing the expressed polypeptide to be detected or purified is also contemplated.
Like the first TAP PCR reaction, the second TAP PCR reaction can be run for any desired number of cycles. In one embodiment, the second TAP PCR reaction is run for about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 cycles or more. Furthermore, any type of thermally stable polymerase can be used for the second TAP PCR reaction. In a particular embodiment the polymerase can be Taq. In some embodiments Vent, Pfu, Tfl, Tth, and Tgo polymerases can be used. The resulting TAP Express PCR fragments from the second PCR reaction can be cleaned by any kit, method or system. A particular kit that can be used to clean the resulting TAP fragments is a Millipore Montage 96-well cleanup kit. Additionally, as discussed above, the level of PCR product recovered can be determined using any detection agent, for example, Pico-Green.
The resulting TAP fragments can be expressed by using any method of gene expression. In one embodiment, the TAP fragments can be expressed using in vivo or in vitro (e.g. cell-free) systems. For example, the fragments can be directly transfected into any eukaryotic or prokaryotic cell for expression. Examples of eukaryotic cells that can be used for expression include mammalian, insect, yeast, and the like. An example of a prokaryotic cell expression system includes E. Coli. The TAP fragments can also be expressed by a cell-free system. According to one embodiment of the invention, the resulting TAP fragments can be expressed in a high-throughput cell-free expression machine, such as, for example, the Roche RTS (Rapid Translation System)-100. In a further embodiment, the TAP fragments can be incubated in Roche RTS100 system at 30° C. for 5 hours. A person with skill in the art can readily appreciate the utility in following the particular cell-free translation machine's instructions. If a T7-histadine promoter or terminator fragment is added to a primary transcript, translation of the TAP fragment can result in histidine tagged polypeptides, which can be purified as discussed below. As discussed herein, any tag can be used.
The expressed Mtb polypeptides can be purified using any purification method for purifying expressed polypeptides. In one embodiment histidine tagged polypeptides can be purified with Qiagen nickel columns, such as Ni-NTA Superflow 96 Biorobot Kit. A person with skill in the art can readily appreciate the utility in following the instructions of the particular polypeptide purification system. Other methods that can be used to purify polypeptides include ultrafiltration, extraction, and chromatography.
The identity, quantity and purity of the purified Mtb polypeptides can be verified by SDS gel electrophoresis. According to one embodiment of the invention, MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) can be employed to confirm the fidelity of the purified polypeptides. According to this embodiment, aliquots of each polypeptide (1-2μg) can be aliquoted into about 48, about 96, about 144, about 192, about 240, about 288, about 336, about 384, about 432, about 480, about 576, about 672, about 768, about 864, about 960, about 1056, about 1152, about 1248, about 1344, about 1440, about 1536 or more well plates and digested with modified trypsin. The resulting material can be mixed with matrix (alpha-cyano-4-hydroxycinnamic acid (CHCA)) and spotted onto any target plate with a suitable number of spots, for example, 48, about 96, about 144, about 192, about 240, about 288, about 336, about 384, about 432, about 480, about 576, about 672, about 768, about 864, about 960, about 1056, about 1152, about 1248, about 1344, about 1440, about 1536 or more spots. In one embodiment, a 384-spot "anchor chip" target plate (Bruker Daltonics, Billerica, Mass.) can be used. The plate can be transferred to the sample stage of a Bruker Autoflex MALDI-TOF mass spectrometer. The spectrometer can be set up to automatically scan the plate and search the Mascot polypeptide database via the Internet. Accordingly, a very rapid verification system can verify purity, identity, and quantity in less than a day, for example, depending on the amount of polypeptides. Purified Mtb polypeptides can be placed in libraries or organized into arrays for subsequent testing and analysis.
Humoral Immune Response
Use of the Mtb polypeptide libraries and arrays prepared, for example, 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 of the present invention, highly immunogenic Mtb antigens are 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
Use of the TAP-expressed Mtb polypeptide libraries and arrays prepared according the methods above (e.g. using TAP or adapter technology) 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 WIC 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 WIC molecules on their membranes to present antigens to T-cells. Efficient T-cell function relies on proper recognition of the WIC-antigen complex. There are two types of WIC molecules: Class I and Class II. The two different classes of WIC 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 present invention, 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 WIC molecules. CD4.sup.+ T-Cells, which includes inflammatory CD4 T-cells and helper CD4 T-cells, bind epitopes that are part of class II MI-IC 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 of the present invention 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 of the invention, a transcriptionally active gene 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.
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. Furthermore, the results of FIG. 5 demonstrate that dendritic cells can present antigens to T-cells supplied from an immunized host after antigenic polypeptides were delivered to the dendritic cells with reagents from the above mentioned applications.
In certain embodiments of the invention, 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 WIC-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 WIC-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-y 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).
Developing a Subunit Vaccine, Pharmaceutical Composition, or Immunogenic Composition
A particular Mtb polypeptide that has been identified to elicit a humoral or cell-mediated immune response, can be further explored to determine its ability to be used in a subunit vaccine, pharmaceutical composition, or immunogenic composition. The terms "subunit vaccine," "DNA vaccine," "recombinant vaccine" and "immunogenic composition" encompass vaccines that are comprised of polypeptides, nucleic acids or a combination of both. Further exploration of a Mtb polypeptide vaccine candidate includes testing the Mtb polypeptide or nucleic acid encoding the Mtb polypeptide in a large number of animal subjects, volunteers or patients. In a particular embodiment, surface antigens can be studied closely because of the likelihood that they can inhibit virus infectivity. In one embodiment, every polypeptide encoded by the Mtb genome is assayed to determine its immunogenic effect. Polypeptides that elicit an immune response, whether cell-mediated or humoral, can be more closely studied to determine potential use alone or in conjunction with other polypeptides and genes as a subunit vaccine, pharmaceutical composition, or immunogenic composition. Suitable methodologies for electing and detecting an immune response are well established in the art.
Uses of Vaccine Compositions
As noted previously, the present invention provides peptide immunogens and nucleic acids encoding the immunogens. As such, the present invention also provides methods of using the immunogens to generate an immune response in a mammalian host.
Methods of generating immune responses in a host are known in the art. However, according to the present invention, the method includes administering to the host an immunogenic composition. The immunogenic composition includes at least one nucleic acid selected from SEQ ID NO: 46-64 and/or 110-121. In addition, fragments of these sequences can be used. Also, it should be noted that combinations of these sequences may be used to generate an immune response against Mtb. When using nucleic acids to generate an immune response the nucleic acids preferably encode peptides found in SEQ ID NO: 65-83 and/or 122-133. In addition, fragments of these sequences can be used. Also, combinations of these sequences can be used.
When combinations of the above immunogenic compositions are to be used at least 2, 3, 4 or 5 or more of the nucleic acids or fragments thereof can be combined to generate an immunogenic composition. Any combination of the nucleic acids finds use in this method.
Also, methods of generating an immune response include administering to the host at least one peptide selected from the peptides found in SEQ ID NO: 65-83 and/or 122-133. In addition, fragments of these sequences can be used. Also, it should be noted that combinations of these sequences may be used to generate an immune response against Mtb. When combinations of the above immunogenic compositions are to be used at least 2, 3, 4 or 5 or more of the nucleic acids or fragments thereof can be combined to generate an immunogenic composition. Any combination of screened nucleic acids finds use in this method.
Various nucleic acids and peptides have been identified that generate an immune response. As such, the nucleic acids and peptides find use in kits. The kits of the invention are useful for a variety of applications including combining reagents necessary for producing vaccine compositions. 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, 5, 10, 15, 20, 25, 30 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 vaccines.
In addition, the kits of the invention 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, 5, 10, 15 or 20 or more peptides for diagnosis of Mtb infection or exposure of an organism, animal or patient.
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.
Procedure for Generating Histidine Tagged TAP Express Fragments
A detailed procedure that is used to produce tagged T7-TAP Express fragments is as follows: 96 different genes were amplified from a mixture of plasmid templates. A first PCR reaction was run with 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 reaction temperature and times for the first PCR reaction were: 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 was added for each kb).
After the first PCR reaction was performed, an aliquot of each PCR reaction from the previous step was transferred into a PCR reaction containing the T7-histidine promoter fragment and T7 terminator fragment. The T7 promoter primer contained 25 bases, while the T7-promoter-His tag fragment contained a 104 base EcoRV/BglII fragment. The T7-terminator fragment was a 74 base oligonucleotide. The reaction temperature and times for the second PCR reaction were: 94° C. for 2 minutes, followed by 30 cycles of: 94° C. for 20 seconds 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).
Using the Mtb Proteome to Identify the Antigenic Targets of Humoral Immunity in Mtb Mice and Humans
The following is a method used to systematically screen and identify antigens in Mtb that give rise to a protective humoral immune response. 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 by were selected for TAP amplification. Initially, three hundred Mtb genes were synthesized by TAP and ˜100 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 ˜100 Mtb polypeptides containing the His tag.
A detailed procedure that was used to produce tagged T7-TAP Express fragments is as follows: groups of 96 Mtb polynucleotide sequences were amplified from Mtb genomic DNA. A first PCR reaction was performed using customized 5' and 3' primers, as shown in Table 1 (SEQ ID NOS: 8 and 9; 10 and 11; 12 and 13; 14 and 15; 16 and 17; 18 and 19; 20 and 21; 22 and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; 32 and 33; 34 and 35; 36 and 37; 38 and 39; 40 and 41; 42 and 43; 44 and 45; 86 and 87; 88 and 89; 90 and 91; 92 and 93; 94 and 95; 96 and 97; 98 and 99; 100 and 101; 102 and 103; 104 and 105; 106 and 107; 108 and 109). 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.
TABLE-US-00001 TABLE 1 Immunogenic Mtb Polypeptides: Primers, Poly- nucleotide Sequences, and Amino Acid Sequences All polynucleotide sequences are shown in the 5' to 3' orientation Rv2031c HEAT SHOCK PROTEIN HSPX (ALPHA-CRSTALLIN HOMOLOG) 14 kDa ANTIGEN) (HSP16.3) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCCACCACCCTT (SEQ ID NO: 8) 3' primer: TGATGATGAGAACCCCCCCCGTTGGTGGACCGGATCTGAA (SEQ ID NO: 9) Polynucleotide sequence: ATGGCCACCACCCTTCCCGTTCAGCGCCACCCGCGGTCCCTCTTCCCCGA GTTTTCTGAGCTGTTCGCGGCCTTCCCGTCATTCGCCGGACTCCGGCCCA CCTTCGACACCCGGTTGATGCGGCTGGAAGACGAGATGAAAGAGGGGCGC TACGAGGTACGCGCGGAGCTTCCCGGGGTCGACCCCGACAAGGACGTCGA CATTATGGTCCGCGATGGTCAGCTGACCATCAAGGCCGAGCGCACCGAGC AGAAGGACTTCGACGGTCGCTCGGAATTCGCGTACGGTTCCTTCGTTCGC ACGGTGTCGCTGCCGGTAGGTGCTGACGAGGACGACATTAAGGCCACCTA CGACAAGGGCATTCTTACTGTGTCGGTGGCGGTTTCGGAAGGGAAGCCAA CCGAAAAGCACATTCAGATCCGGTCCACCAAC 435 bp (SEQ ID NO: 46) Amino acid sequence: MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGR YEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVR TVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN (SEQ ID NO: 65) RV3763 19 KDA LIPOPROTEIN ANTIGEN PRECURSOR LPQH 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATGTGAAGCGTGGACTG (SEQ ID NO: 10) 3' primer: TGATGATGAGAACCCCCCCCGGAACAGGTCACCTCGATTT (SEQ ID NO: 11) Polynucleotide sequence: GTGAAGCGTGGACTGACGGTCGCGGTAGCCGGAGCCGCCATTCTGGTCGC AGGTCTTTCCGGATGTTCAAGCAACAAGTCGACTACAGGAAGCGGTGAGA CCACGACCGCGGCAGGCACGACGGCAAGCCCCGGCGCCGCCTCCGGGCCG AAGGTCGTCATCGACGGTAAGGACCAGAACGTCACCGGCTCCGTGGTGTG CACAACCGCGGCCGGCAATGTCAACATCGCGATCGGCGGGGCGGCGACCG GCATTGCCGCCGTGCTCACCGACGGCAACCCTCCGGAGGTGAAGTCCGTT GGGCTCGGTAACGTCAACGGCGTCACGCTGGGATACACGTCGGGCACCGG ACAGGGTAACGCCTCGGCAACCAAGGACGGCAGCCACTACAAGATCACTG GGACCGCTACCGGGGTCGACATGGCCAACCCGATGTCACCGGTGAACAAG TCGTTCGAAATCGAGGTGACCTGTTCC 480 bp (SEQ ID NO: 47) Amino acid sequence: VKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGAASGP KVVIDGKDQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSV GLGNVNGVTLGYTSGTGQGNASATKDGSHYKITGTATGVDMANPMSPVNK SFEIEVTCS (SEQ ID NO: 66) Rv2744c CONSERVED 35 KDA ALANINE RICH PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCCAATCCGTTC (SEQ ID NO: 12) 3' primer: TGATGATGAGAACCCCCCCCCTGACCGTAGGGCTGCTCGG (SEQ ID NO: 13) Polynucleotide sequence: ATGGCCAATCCGTTCGTTAAAGCCTGGAAGTACCTCATGGCGCTGTTCAG CTCGAAGATCGACGAGCATGCCGACCCCAAGGTGCAGATTCAACAGGCCA TTGAGGAAGCACAGCGCACCCACCAAGCGCTGACTCAACAGGCGGCGCAA GTGATCGGTAACCAGCGTCAATTGGAGATGCGACTCAACCGACAGCTGGC GGACATCGAAAAGCTTCAGGTCAATGTGCGCCAAGCCCTGACGCTGGCCG ACCAGGCCACCGCCGCCGGAGACGCTGCCAAGGCCACCGAATACAACAAC GCCGCCGAGGCGTTCGCAGCCCAGCTGGTGACCGCCGAGCAGAGCGTCGA AGACCTCAAGACGCTGCATGACCAGGCGCTTAGCGCCGCAGCTCAGGCCA AGAAGGCCGTCGAACGAAATGCGATGGTGCTGCAGCAGAAGATCGCCGAG CGAACCAAGCTGCTCAGCCAGCTCGAGCAGGCGAAGATGCAGGAGCAGGT CAGCGCATCGTTGCGGTCGATGAGTGAGCTCGCCGCGCCAGGCAACACGC CGAGCCTCGACGAGGTGCGCGACAAGATCGAGCGTCGCTACGCCAACGCG ATCGGTTCGGCTGAACTTGCCGAGAGTTCGGTGCAGGGCCGGATGCTCGA GGTGGAGCAGGCCGGGATCCAGATGGCCGGTCATTCACGGTTGGAACAGA TCCGCGCATCGATGCGCGGTGAAGCGTTGCCGGCCGGCGGGACCACGGCT ACCCCCAGACCGGCCACCGAGACTTCTGGCGGGGCTATTGCCGAGCAGCC CTACGGTCAG 813 bp (SEQ ID NO: 48) Amino acid sequence: MANPFVKAWKYLMALFSSKIDEHADPKVQIQQAIEEAQRTHQALTQQAAQ VIGNQRQLEMRLNRQLADIEKLQVNVRQALTLADQATAAGDAAKATEYNN AAEAFAAQLVTAEQSVEDLKTLHDQALSAAAQAKKAVERNAMVLQQKIAE RTKLLSQLEQAKMQEQVSASLRSMSELAAPGNTPSLDEVRDKIERRYANA IGSAELAESSVQGRMLEVEQAGIQMAGHSRLEQIPASMRGEALPAGGTTA TPRPATETSGGAIAEQPYGQ (SEQ ID NO: 67) Rv0097 POSSIBLE OXIDOREDUCTASE 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGACGCTTAAGGTC (SEQ ID NO: 14) 3' primer: TGATGATGAGAACCCCCCCCTGCCGCGTATCCCGGCGTCT (SEQ ID NO: 15) Polynucleotide sequence: ATGACGCTTAAGGTCAAAGGCGAGGGACTCGGTGCGCAGGTCACAGGGGT CGATCCCAAGAATCTGGACGATATAACCACCGACGAGATCCGGGATATCG TTTACACGAACAAGCTCGTTGTGCTAAAAGACGTCCATCCGTCTCCGCGG GAGTTCATCAAACTCGGCAGGATAATTGGACAAATCGTTCCGTATTACGA ACCCATGTACCATCACGAAGACCACCCGGAGATCTTTGTCTCCTCCACTG AGGAAGGTCAGGGGGTCCCAAAAACCGGCGCGTTCTGGCATATCGACTAT ATGTTTATGCCGGAACCTTTCGCGTTTTCCATGGTGCTGCCGCTGGCGGT GCCTGGACACGACCGCGGGACCTATTTCATCGATCTCGCCAGGGTCTGGC AGTCGCTGCCCGCCGCCAAGCGAGACCCGGCCCGCGGAACCGTCAGCACC CACGACCCTCGACGCCACATCAAGATCCGACCCAGCGACGTCTACCGGCC CATCGGAGAGGTATGGGACGAGATCAACCGGACCACGCCCCCAATAAAGT GGCCTACGGTCATCCGGCACCCAAAGACCGGCCAAGAGATCCTCTACATC TGCGCGACGGGCACCACCAAGATCGAGGACAAGGACGGCAATCCGGTTGA TCCGGAGGTGCTGCAAGAACTCATGGCCGCGACCGGACAGCTCGATCCTG AGTACCAGTCGCCGTTCATACATACTCAGCACTACCAGGTTGGCGACATC ATCTTGTGGGACAACCGGGTTCTCATGCACCGAGCGAAGCACGGCAGCGC CGCGGGCACTCTGACGACCTACCGCCTGACCATGCTTGATGGCCTCAAGA CGCCGGGATACGCGGCA 870 (SEQ ID NO: 49) Amino acid sequence: MTLKVKGEGLGAQVTGVDPKNLDDITTDEIRDIVYTNKLVVLKDVHPSPR EFIKLGRIIGQIVPYYEPMYHHEDHPEIFVSSTEEGQGVPKTGAFWHIDY MFMPEPFAFSMVLPLAVPGHDRGTYFIDLARVWQSLPAAKRDPARGTVST HDPRRHIKIRPSDVYRPIGEVWDEINRTTPPIKWPTVIRHPKTGQEILYI CATGTTKIEDKDGNPVDPEVLQELMAATGQLDPEYQSPFIHTQHYQVGDI ILWDNRVLMHRAKHGSAAGTLTTYRLTMLDGLKTPGYAA (SEQ ID NO: 68) Rv0475 IRON-REGULATED HEPARIN BINDING HEMAGGLUTI- NIN HBHA (ADHESIN) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCTGAAAACTCG (SEQ ID NO: 16) 3' primer: TGATGATGAGAACCCCCCCCCTTCTGGGTGACCTTCTTGG (SEQ ID NO: 17) Polynucleotide sequence: ATGGCTGAAAACTCGAACATTGATGACATCAAGGCTCCGTTGCTTGCCGC GCTTGGAGCGGCCGACCTGGCCTTGGCCACTGTCAACGAGTTGATCACGA ACCTGCGTGAGCGTGCGGAGGAGACTCGTACGGACACCCGCAGCCGGGTC GAGGAGAGCCGTGCTCGCCTGACCAAGCTGCAGGAAGATCTGCCCGAGCA GCTCACCGAGCTGCGTGAGAAGTTCACCGCCGAGGAGCTGCGTAAGGCCG CCGAGGGCTACCTCGAGGCCGCGACTAGCCGGTACAACGAGCTGGTCGAG CGCGGTGAGGCCGCTCTAGAGCGGCTGCGCAGCCAGCAGAGCTTCGAGGA AGTGTCGGCGCGCGCCGAAGGCTACGTGGACCAGGCGGTGGAGTTGACCC AGGAGGCGTTGGGTACGGTCGCATCGCAGACCCGCGCGGTCGGTGAGCGT GCCGCCAAGCTGGTCGGCATCGAGCTGCCTAAGAAGGCTGCTCCGGCCAA GAAGGCCGCTCCGGCCAAGAAGGCCGCTCCGGCCAAGAAGGCGGCGGCCA AGAAGGCGCCCGCGAAGAAGGCGGCGGCCAAGAAGGTCACCCAGAAG 600 bp (SEQ ID NO: 50) Amino acid sequence: MAENSNIDDIKAPLLAALGAADLALATVNELITNLRERAEETRTDTRSRV EESRARLTKLQEDLPEQLTELREKFTAEELRKAAEGYLEAATSRYNELVE RGEAALERLRSQQSFEEVSARAEGYVDQAVELTQEALGTVASQTPAVGEP AAKLVGIELPKKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQK (SEQ ID NO: 69) Rv3117 PROBABLE THIOSULFATE SULFURTRANSFERASE CYSA3 (RHODANESE-LIKE PROTEIN) (THIOSULFATE CYANIDE TRANSSULFURASE) (THIOSULFATE THIOTRANSFERASE) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCACGCTGCGAT (SEQ ID NO: 18) 3' primer: TGATGATGAGAACCCCCCCCGCTTCCCAACTCGATCGGGG (SEQ ID NO: 19) Polynucleotide sequence: ATGGCACGCTGCGATGTCCTGGTCTCCGCCGACTGGGCTGAGAGCAATCT GCACGCGCCGAAGGTCGTTTTCGTCGAAGTGGACGAGGACACCAGTGCAT ATGACCGTGACCATATTGCCGGCGCGATCAAGTTGGACTGGCGCACCGAC CTGCAGGATCCGGTCAAACGTGACTTCGTCGACGCCCAGCAATTCTCCAA GCTGCTGTCCGAGCGTGGCATCGCCAACGAGGACACGGTGATCCTGTACG GCGGCAACAACAATTGGTTCGCCGCCTACGCGTACTGGTATTTCAAGCTC TACGGCCATGAGAAGGTCAAGTTGCTCGACGGCGGCCGCAAGAAGTGGGA GCTCGACGGACGCCCGCTGTCCAGCGACCCGGTCAGCCGGCCGGTGACCT CCTACACCGCCTCCCCGCCGGATAACACGATTCGGGCATTCCGCGACGAG GTCCTGGCGGCCATCAACGTCAAGAACCTCATCGACGTGCGCTCTCCCGA CGAGTTCTCCGGCAAGATCCTGGCCCCCGCGCACCTGCCGCAGGAACAAA GCCAGCGGCCCGGACACATTCCTGGTGCCATCAACGTGCCGTGGAGCAGG GCCGCCAACGAGGACGGCACCTTCAAGTCCGATGAGGAGTTGGCCAAGCT TTACGCCGACGCCGGCCTAGACAACAGCAAGGAAACGATTGCCTACTGCC GAATCGGGGAACGGTCCTCGCACACCTGGTTCGTGTTGCGGGAATTACTC GGACACCAAAACGTCAAGAACTACGACGGCAGTTGGACAGAATACGGCTC CCTGGTGGGCGCCCCGATCGAGTTGGGAAGC 834 bp (SEQ ID NO: 51) Amino acid sequence: MARCDVLVSADWAESNLHAPKVVFVEVDEDTSAYDRDHIAGAIKLDWRTD LQDPVKRDFVDAQQFSKLLSERGIANEDTVILYGGNNNWFAAYAYWYFKL YGHEKVKLLDGGRKKWELDGRPLSSDPVSRPVTSYTASPPDNTIRAFRDE VLAAINVKNLIDVRSPDEFSGKILAPAHLPQEQSQRPGHIPGAINVPWSR AANEDGTFKSDEELAKLYADAGLDNSKETIAYCRIGERSSHTWFVLRELL GHQNVKNYDGSWTEYGSLVGAPIELGS (SEQ ID NO: 70) Rv1347c CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGACCAAACCCACA (SEQ ID NO: 20) 3' primer: TGATGATGAGAACCCCCCCCCGCAGCCGTGGTCGGAGCTT (SEQ ID NO: 21) Polynucleotide sequence: ATGACCAAACCCACATCCGCTGGCCAGGCCGACGACGCGCTGGTTCGGCT AGCCCGCGAGCGATTCGACCTACCTGACCAGGTACGACGCCTCGCCCGCC CGCCCGTTCCATCGTTGGAGCCGCCATACGGGTTGCGGGTCGCACAGCTG ACCGACGCGGAGATGTTGGCGGAGTGGATGAACCGTCCTCATCTGGCGGC GGCCTGGGAGTACGACTGGCCGGCGTCACGTTGGCGTCAACACCTGAACG CCCAACTTGAGGGAACCTATTCGTTGCCATTGATCGGCAGCTGGCACGGA ACAGATGGTGGTTATCTCGAATTATACTGGGCAGCAAAGGATTTGATTTC TCACTACTACGACGCAGACCCCTACGATTTGGGGCTGCACGCGGCCATCG CGGACTTGTCGAAGGTCAATCGGGGCTTCGGCCCGCTGCTGCTACCGCGG ATCGTGGCCAGCGTCTTTGCCAACGAGCCGCGTTGCCGGCGGATCATGTT CGACCCCGATCACCGCAACACCGCGACCCGTCGGTTGTGTGAGTGGGCCG GATGCAAGTTCCTCGGTGAGCATGACACGACAAACCGGCGCATGGCGCTC TACGCTTTGGAAGCTCCGACCACGGCTGCG 633 bp (SEQ ID NO: 52) Amino acid sequence: MTKPTSAGQADDALVRLARERFDLPDQVRRLARPPVPSLEPPYGLRVAQL TDAEMLAEWMNRPHLAAAWEYDWPASRWRQHLNAQLEGTYSLPLIGSWHG TDGGYLELYWAAKDLISHYYDADPYDLGLHAAIADLSKVNRGFGPLLLPR IVASVFANEPRCRRIMFDPDHRNTATRRLCEWAGCKFLGEHDTTNRRMAL YALEAPTTAA (SEQ ID NO: 71) Rv0815c PROBABLE THIOSULFATE SULFURTRANSFERASE CYSA2 (RHODANESE-LIKE PROTEIN) (THIOSULFATE CYA-
NIDE TRANSSULFURASE) (THIOSULFATE THIOTRANSFERASE) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCACGCTGCGAT (SEQ ID NO: 22) 3' primer: TGATGATGAGAACCCCCCCCGCTTCCCAACTCGATCGGGG (SEQ ID NO: 23) Polynucleotide sequence: ATGGCACGCTGCGATGTCCTGGTCTCCGCCGACTGGGCTGAGAGCAATCT GCACGCGCCGAAGGTCGTTTTCGTCGAAGTGGACGAGGACACCAGTGCAT ATGACCGTGACCATATTGCCGGCGCGATCAAGTTGGACTGGCGCACCGAC CTGCAGGATCCGGTCAAACGTGACTTCGTCGACGCCCAGCAATTCTCCAA GCTGCTGTCCGAGCGTGGCATCGCCAACGAGGACACGGTGATCCTGTACG GCGGCAACAACAATTGGTTCGCCGCCTACGCGTACTGGTATTTCAAGCTC TACGGCCATGAGAAGGTCAAGTTGCTCGACGGCGGCCGCAAGAAGTGGGA GCTCGACGGACGCCCGCTGTCCAGCGACCCGGTCAGCCGGCCGGTGACCT CCTACACCGCCTCCCCGCCGGATAACACGATTCGGGCATTCCGCGACGAG GTCCTGGCGGCCATCAACGTCAAGAACCTCATCGACGTGCGCTCTCCCGA CGAGTTCTCCGGCAAGATCCTGGCCCCCGCGCACCTGCCGCAGGAACAAA GCCAGCGGCCCGGACACATTCCTGGTGCCATCAACGTGCCGTGGAGCAGG GCCGCCAACGAGGACGGCACCTTCAAGTCCGATGAGGAGTTGGCCAAGCT TTACGCCGACGCCGGCCTAGACAACAGCAAGGAAACGATTGCCTACTGCC GAATCGGGGAACGGTCCTCGCACACCTGGTTCGTGTTGCGGGAATTACTC GGACACCAAAACGTCAAGAACTACGACGGCAGTTGGACAGAATACGGCTC CCTGGTGGGCGCCCCGATCGAGTTGGGAAGC 834 bp (SEQ ID NO: 53) Amino acid sequence: MARCDVLVSADWAESNLHAPKVVFVEVDEDTSAYDRDHIAGAIKLDWRTD LQDPVKRDFVDAQQFSKLLSERGIANEDTVILYGGNNNWFAAYAYWYFKL YGHEKVKLLDGGRKKWELDGRPLSSDPVSRPVTSYTASPPDNTIRAFRDE VLAAINVKNLIDVRSPDEFSGKILAPAHLPQEQSQRPGHIPGAINVPWSR AANEDGTFKSDEELAKLYADAGLDNSKETIAYCRIGERSSHTWFVLRELL GHQNVKNYDGSWTEYGSLVGAPIELGS (SEQ ID NO: 72) Rv2613c CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATGTGAGTGACGAGGAC (SEQ ID NO: 24) 3' primer: TGATGATGAGAACCCCCCCCTGGTTGCCGAGCCCACTCGG (SEQ ID NO: 25) Polynucleotide sequence: GTGAGTGACGAGGACCGCACGGATCGGGCCACCGAGGACCACACCATCTT CGATCGGGGTGTCGGCCAGCGCGACCAGCTGCAGCGGTTATGGACCCCCT ACCGGATGAACTACCTGGCCGAAGCGCCAGTGAAGCGTGACCCCAATTCC TCGGCCAGCCCTGCGCAGCCGTTCACCGAGATCCCGCAGCTGTCCGACGA AGAGGGTCTGGTGGTCGCTCGTGGCAAGCTGGTCTACGCCGTGCTCAACC TGTACCCGTACAACCCCGGGCACTTGATGGTGGTGCCCTATCGTCGGGTA TCCGAACTCGAGGATCTCACCGATTTGGAGAGCGCCGAGTTGATGGCGTT CACCCAGAAGGCGATTCGCGTGATCAAGAACGTGTCGCGTCCGCACGGCT TCAATGTCGGCCTGAACCTAGGGACATCGGCGGGCGGGTCGCTGGCCGAG CACCTGCACGTGCATGTGGTGCCACGGTGGGGTGGCGATGCGAATTTCAT CACCATCATCGGGGGCTCCAAGGTGATTCCGCAGCTGCTGCGCGACACCC GTCGGCTGCTTGCCACCGAGTGGGCTCGGCAACCA 588 bp (SEQ ID NO: 54) Amino acid sequence: VSDEDRTDRATEDHTIFDRGVGQRDQLQRLWTPYRMNYLAEAPVKRDPNS SASPAQPFTEIPQLSDEEGLVVARGKLVYAVLNLYPYNPGHLMVVPYRRV SELEDLTDLESAELMAFTQKAIRVIKNVSRPHGFNVGLNLGTSAGGSLAE HLHVHVVPRWGGDANFITIIGGSKVIPQLLRDTRRLLATEWARQP (SEQ ID NO: 73) Rv3226c CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGTGCGGACGGTTT (SEQ ID NO: 26) 3' primer: TGATGATGAGAACCCCCCCCCAGCAGCTGGATCTGCTCGG (SEQ ID NO: 27) Polynucleotide sequence: ATGTGCGGACGGTTTGCGGTCACCACTGATCCGGCCCAGCTGGCCGAGAA AATCACGGCCATAGACGAGGCCACCGGGTGCGGTGGCGGGAAGACGAGCT ACAACGTGGCACCCACCGACACGATCGCGACAGTGGTGTCCCGCCACAGC GAGCCCGACGACGAGCCCACCCGCCGGGTGCGGCTCATGCGCTGGGGACT GATTCCGTCGTGGATCAAGGCCGGGCCCGGCGGCGCACCCGATGCCAAAG GCCCACCGCTGATCAACGCCCGCGCCGATAAGGTCGCCACGTCGCCGGCG TTCCGGAGTGCGGTCAGAAGTAAGCGTTGCCTGGTGCCGATGGACGGCTG GTACGAATGGCGCGTCGACCCCGACGCCACCCCGGGGAGGCCGAACGCCA AGACGCCGTTCTTCCTGCACCGCCACGACGGCGCCCTGTTGTTCACGGCC GGGCTGTGGTCGGTTTGGAAGTCTTACAGGTCCGCCCCACCGCTGCTGAG CTGCACGGTGATCACCACCGATGCCGTGGGCGAGCTGGCCGAGATCCATG ACCGGATGCCGCTGCTGCTGGCCGAAGAGGACTGGGACGACTGGCTGAAT CCAGACGCCCCGCCGGATCCTGAGCTGCTGGCCCGCCCGCCGGATGTGCG CGACATCGCGCTGCGCCAAGTGTCCACGTTGGTCAACAACGTGCGCAACA ACGGGCCTGAGCTGTTGGAGCCGGCCAGGTCGCAGCCCGAGCAGATCCAG CTGCTG 759 bp (SEQ ID NO: 55) Amino acid sequence: MCGRFAVTTDPAQLAEKITAIDEATGCGGGKTSYNVAPTDTIATVVSRHS EPDDEPTRRVRLMRWGLIPSWIKAGPGGAPDAKGPPLINARADKVATSPA FRSAVRSKRCLVPMDGWYEWRVDPDATPGRPNAKTPFFLHRHDGALLFTA GLWSVWKSYRSAPPLLSCTVITTDAVGELAEIHDRMPLLLAEEDWDDWLN PDAPPDPELLARPPDVRDIALRQVSTLVNNVRNNGPELLEPARSQPEQIQ LL (SEQ ID NO: 74) Rv0349 HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATGTGCCAGAGCTGGAG (SEQ ID NO: 28) 3' primer: TGATGATGAGAACCCCCCCCGTCCGCCAGCTTGACCGACT (SEQ ID NO: 29) Polynucleotide sequence: GTGCCAGAGCTGGAGACGCCCGACGACCCAGAGTCGATATACCTTGCCCG CCTCGAGGATGTCGGAGAACACAGACCGACGTTCACGGGCGACATCTACC GACTCGGCGATGGTCGCATGGTGATGATCCTCCAGCACCCATGCGCGCTG CGGCACGGCGTTGACCTCCATCCGCGACTGCTGGTCGCTCCCGTAAGACC CGACTCGCTTCGTTCCAACTGGGCTAGAGCCCCGTTCGGCACGATGCCGC TTCCGAAGCTCATCGACGGTCAGGATCACTCGGCGGACTTCATCAATCTT GAACTCATCGATTCACCAACGCTTCCGACCTGTGAGCGGATCGCGGTGCT CAGCCAGTCAGGCGTCAACTTGGTCATGCAACGGTGGGTGTACCACAGCA CCCGGCTCGCCGTGCCCACGCACACCTACTCCGACAGCACCGTTGGCCCG TTCGATGAGGCAGACCTGATCGAGGAGTGGGTGACGGATCGCGTCGACGA TGGGGCCGACCCGCAGGCGGCCGAACACGAATGCGCCTCCTGGCTCGATG AAAGAATCAGCGGCCGCACTCGGCGAGCGCTGCTCAGCGACCGTCAGCAC GCCAGTTCAATACGGCGAGAAGCGCGTTCTCATCGAAAGTCGGTCAAGCT GGCGGAC 660 bp (SEQ ID NO: 56) Amino acid sequence: VPELETPDDPESIYLARLEDVGEHRPTFTGDIYRLGDGRMVMILQHPCAL RHGVDLHPRLLVAPVRPDSLRSNWARAPFGTMPLPKLIDGQDHSADFINL ELIDSPTLPTCERIAVLSQSGVNLVMQRWVYHSTRLAVPTHTYSDSTVGP FDEADLIEEWVTDRVDDGADPQAAEHECASWLDERISGRTRPALLSDRQH ASSIRREARSHRKSVKLAD (SEQ ID NO: 75) Rv0009 PROBABLE IRON-REGULATED PEPTIDYL-PROLYL CIS-TRANS ISOMERASE A PPIA (PPIase A) (ROTAMASE A) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCAGACTGTGAT (SEQ ID NO: 30) 3' primer: TGATGATGAGAACCCCCCCCGGAGATGGTGATCGACTCGA (SEQ ID NO: 31) Polynucleotide sequence: ATGGCAGACTGTGATTCCGTGACTAACAGCCCCCTTGCGACCGCTACCGC CACGCTGCACACTAACCQCGGCGACATCAAGATCGCCCTGTTCGGAAACC ATGCGCCCAAGACCGTCGCCAATTTTGTGGGCCTTGCGCAGGGCACCAAG GACTATTCGACCCAAAACGCATCAGGTGGCCCGTCCGGCCCGTTCTACGA CGGCGCGGTCTTTCACCGGGTGATCCAGGGCTTCATGATCCAGGGTGGCG ATCCAACCGGGACGGGTCGCGGCGGACCCGGCTACAAGTTCGCCGACGAG TTCCACCCCGAGCTGCAATTCGACAAGCCCTATCTGCTCGCGATGGCCAA CGCCGGTCCGGGCACCAACGGCTCACAGTTTTTCATCACCGTCGGCAAGA CTCCGCACCTGAACCGGCGCCACACCATTTTCGGTGAAGTGATCGACGCG GAGTCACAGCGGGTTGTGGAGGCGATCTCCAAGACGGCCACCGACGGCAA CGATCGGCCGACGGACCCGGTGGTGATCGAGTCGATCACCATCTCC 549 bp (SEQ ID NO: 57) Amino acid sequence: MADCDSVTNSPLATATATLHTNRGDIKIALFGNHAPKTVANFVGLAQGTK DYSTQNASGGPSGPFYDGAVFHRVIQGFMIQGGDPTGTGRGGPGYKFADE FHPELQFDKPYLLAMANAGPGTNGSQFFITVGKTPHLNRRHTIFGEVIDA ESQRVVEAISKTATDGNDRPTDPVVIESITIS (SEQ ID NO: 76) Rv1073 CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGGGGCGCAGCCG (SEQ ID NO: 32) 3' primer: TGATGATGAGAACCCCCCCCACGTCGTGATGTCAACGTGT (SEQ ID NO: 33) Polynucleotide sequence: ATGGGGGCGCAGCCGTTCATCGGCAGCGAGGCGTTGGCGGCGGGACTCAT CAGCTGGCATGAGCTGGGCAAGTACTACACCGCGATCATGCCCAACGTCT ATCTGGACAAGCGGCTGAAGCCCTCCCTGCGGCAACGCGTTATCGCGGCC TGGCTGTGGTCGGGCCGCAAAGGGGTGATCGCCGGCGCTTCGGCATCAGC GCTGCACGGCGCGAAATGGGTCGATGACCACGCATTGGTGGAGTTGATCT GGCGCAACGCCAGGGCGCCGAACGGGGTGCGGACTAAGGATGAGCTACTG CTCGACGGCGAAGTCCAGCGCTTGTGCGGGCTTACTGTGACTACCGTTGA ACGTACGGCCTTCGACTTGGGCAGGCGTCCACCCTTAGGTCAGGCGATAA CCAGACTGGATGCGCTTGCCAATGCCACCGATTTCAAGATCAACGATGTT AGGGAGCTCGCGAGGAAGCACCCCCATACTCGCGGGCTGCGTCAACTAGA CAAGGCGCTGGATCTCGTCGACCCAGGTGCGCAGTCGCCGAAGGAGACGT GGCTGCGGCTCTTGCTGATAAACGCCGGCTTTCCACGGCCGTCCACTCAG ATCCCCTTGCTCGGCGTCTACGGGCATCCAAAGTATTTCCTCGACATGGG ATGGGAGGACATCATGCTCGCGGTCGAGTACGACGGCGAGCAACACCGTC TCAGCCGAGACCAGTTCGTCAAAGACGTCGAACGCCTGGAATACATCCGG CGCGCCGGCTGGACTCACATCAGGGTGCTGGCAGACCACAAGGGACCCGA CGTCGTCCGCCGGGTTCGGCAGGCTTGGGACACGTTGACATCACGACGT 852 bp (SEQ ID NO: 58) Amino acid sequence: MGAQPFIGSEALAAGLISWHELGKYYTAIMPNVYLDKRLKPSLRQRVIAA WLWSGRKGVIAGASASALHGAKWVDDHALVELIWRNARAPNGVRTKDELL LDGEVQRLCGLTVTTVERTAFDLGRRPPLGQAITRLDALANATDFKINDV RELARKHPHTRGLRQLDKALDLVDPGAQSPKETWLRLLLINAGFPRPSTQ IPLLGVYGHPKYFLDMGWEDIMLAVEYDGEQHRLSRDQFVKDVERLEYIR RAGWTHIRVLADHKGPDVVRRVRQAWDTLTSRR (SEQ ID NO: 77) Rv0781 PROBABLE PROTEASE II PTRBA [FIRST PART] (OLIGOPEPTIDASE B) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGATGCACCGAACC (SEQ ID NO: 34) 3' primer: TGATGATGAGAACCCCCCCCTCGGCTTCGTGGTAAACCCG (SEQ ID NO: 35) Polynucleotide sequence: ATGATGCACCGAACCGCACTACCCTCACCGCCCGTGGCCAAGCGGGTGCA GACCCGC:CGGGAGCACCACGGCGACGTCTTTGTCGACCCATATGAATGG TTGCGCGACAAGGACAGCCCTGAAGTAATCGCCTACCTCGAAGCTGAAAA CGACTACACCGAACGGACCACCGCGCACCTTGAGCCATTGCGGCAAAAGA TCTTCCACGAAATCAAAGCGCGTACCAAGGAAACCGACTTATCGGTGCCG ACGCGACGTGGCAACTGGTGGTACTACGCGCGGACCTTTGAGGGAAAGCA GTATGGCGTACACTGTCGTTGCCCGGTAACCGATCCCGACGACTGGAACC CACCAGAGTTCGACGAGCGCACCGAAATACCCGGTGAACAGCTTCTGCTC GACGAGAACGTGGAAGCTGACGGCCACGACTTCTTCGCACTGGGCGCGGC CAGCGTCAGCCTGGACGATAACCTCTTAGCGTATTCCGTTGATGTCGTAG GTGACGAACGATATACCTTGCGGTTCAAGGATTTACGCACCGGAGAACAG TACCCGGACGAGATCGCCGGGATCGGAGCGGGAGTCACCTGGGCAGCTGA CAACCACTGTCTACTACACCACCGTGGACGCGGCCTGGCGTCCGGACACA GTGTGGCGATACCGACTAGGGTCCGGCGAATCGTCGGAGCGGGTTTACCA CGAAGCCGA 711 bp (SEQ ID NO: 59) Amino acid sequence: MMHRTALPSPPVAKRVQTRREHHGDVFVDPYEWLRDKDSPEVIAYLEAEN DYTERTTAHLEPLRQKIFHEIKARTKETDLSVPTRRGNWWYYARTFEGKQ YGVHCRCPVTDPDDWNPPEFDERTEIPGEQLLLDENVEADGHDFFALGAA SVSLDDNLLAYSVDVVGDERYTLRFKDLRTGEQYPDEIAGIGAGVTWAAD NHCLLHHRGRGLASGHSVAIPTRVRRIVGAGLPRSR (SEQ ID NO: 78) Rv2108 PPE FAMILY PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGCCCAATTTCTGG (SEQ ID NO: 36)
3' primer: TGATGATGAGAACCCCCCCCAAACTTAGGATGTTCCTTGT (SEQ ID NO: 37) Polynucleotide sequence: ATGCCCAATTTCTGGGCGTTGCCGCCCGAGATCAACTCCACCCGGATATA TCTCGGCCCGGGTTCTGGCCCGATACTGGCCGCCGCCCAGGGATGGAACG CTCTGGCCAGTGAGCTGGAAAAGACGAAGGTGGGGTTGCAGTCAGCGCTC GACACGTTGCTGGAGTCGTATAGGGGTCAGTCGTCGCAGGCTTTGATACA GCAGACCTTGCCGTATGTGCAGTGGCTGACCACGACCGCCGAGCACGCCC ATAAGACCGCGATCCAGCTCACGGCAGCGGCGAACGCCTACGAGCAGGCT AGAGCGGCGATGGTGCCGCCGGCGATGGTGCGCGCGAACCGCGTGCAGAC CACAGTGTTGAAGGCAATCAACTGGTTCGGGCAATTCTCCACCAGGATCG CCGACAAGGAGGCCGACTACGAACAGATGTGGTTCCAAGACGCGCTAGTG ATGGAGAACTATTGGGAAGCCGTGCAAGAGGCGATACAGTCGACGTCGCA TTTTGAGGATCCACCGGAGATGGCCGACGACTACGACGAGGCCTGGATGC TCAACACCGTGTTCGACTATCACAACGAGAACGCAAAAGAGGAGGTCATC CATCTCGTGCCCGACGTGAACAAGGAGAGGGGGCCCATCGAACTCGTAAC CAAGGTAGACAAAGAGGGGACCATCAGACTCGTCTACGATGGGGAGCCCA CGTTTTCATACAAGGAACATCCTAAGTTT 732 bp (SEQ ID NO: 60) Amino acid sequence: MPNFWALPPEINSTRIYLGPGSGPILAAAQGWNALASELEKTKVGLQSAL DTLLESYRGQSSQALIQQTLPYVQWLTTTAEHAHKTAIQLTAAANAYEQA RAAMVPPAMVRANRVQTTVLKAINWFGQFSTRIADKEADYEQMWFQDALV MENYWEAVQEAIQSTSHFEDPPEMADDYDEAWMLNTVFDYHNENAKEEVI HLVPDVNKERGPIELVTKVDKEGTIRLVYDGEPTFSYKEHPKF (SEQ ID NO: 79) Rv3920c HYPOTHETICAL PROTEIN SIMILAR TO JAG PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGGCCGACGCTGAC (SEQ ID NO: 38) 3' primer: TGATGATGAGAACCCCCCCCGTCGCGGAGCACAACGACTC (SEQ ID NO: 39) Polynucleotide sequence: ATGGCCGACGCTGACACCACCGACTTCGACGTCGACGCAGAAGCACCGGG TGGAGGCGTCCGGGAGGACACGGCGACGGATGCTGACGAGGCCGACGATC AAGAAGAGAGATTGGTCGCCGAGGGCGAGATTGCAGGCGACTACCTGGAA GAGTTATTGGACGTGTTGGACTTCGATGGCGACATCGACCTCGATGTCGA AGGCAATCGTGCGGTGGTGAGCATCGACGGCAGTGACGACCTGAACAAGT TGGTCGGGCGCGGGGGCGAGGTGCTCGACGCTCTGCAGGAACTCACCCGG TTGGCGGTGCATCAGAAGACCGGTGTGCGGAGCCGGTTGATGCTAGACAT CGCGAGGTGGCGACGGCGGCGCCGGGAGGAATTGGCGGCGCTGGCCGACG AGGTGGCGCGGCGAGTGGCCGAAACCGGTGACCGCGAGGAACTCGTTCCA ATGACGCCGTTCGAACGGAAGATCGTCCACGATGCGGTTGCAGCGGTGCC AGGTGTGCACAGCGAAAGCGAAGGCGTGGAGCCAGAACGCCGAGTCGTTG TGCTCCGCGAC 564 (SEQ ID NO: 61) Amino acid sequence: MADADTTDFDVDAEAPGGGVREDTATDADEADDQEERLVAEGEIAGDYLE ELLDVLDFDGDIDLDVEGNRAVVSIDGSDDLNKLVGRGGEVLDALQELTR LAVHQKTGVRSRLMLDIARWRRRRREELAALADEVARRVAETGDREELVP MTPFERKIVHDAVAAVPGVHSESEGVEPERRVVVLRD (SEQ ID NO: 80) Rv1044 CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATTTGTGTGCAAAACCG (SEQ ID NO: 40) 3' primer: TGATGATGAGAACCCCCCCCCGCCGATGCTCGCTTCGGCC (SEQ ID NO: 41) Polynucleotide sequence: TTGTGTGCAAAACCGTATCTAATTGATACGATTGCGCACATGGCTATCTG GGATCGCCTCGTCGAGGTTGCCGCCGAGCAACATGGCTACGTCACGACTC GCGATGCGCGAGACATCGGCGTCGACCCTGTGCAGCTCCGCCTCCTAGCG GGGCGCGGACGTCTTGAGCGTGTCGGCCGAGGTGTGTACCGGGTGCCCGT GCTGCCGCGTGGTGAGCACGACGATCTCGCAGCCGCAGTGTCGTGGACTT TGGGGCGTGGCGTTATCTCGCATGAGTCGGCCTTGGCGCTTCATGCCCTC GCTGACGTGAACCCGTCGCGCATCCATCTCACCGTCCCGCGCAACAACCA TCCGCGTGCGGCCGGGGGCGAGCTGTACCGAGTTCACCGCCGCGACCTCC AGGCAGCCCACGTCACTTCGGTCGACGGAATACCCGTCACGACGGTTGCG CGCACCATCAAAGACTGCGTGAAGACGGGCACGGATCCTTATCAGCTTCG GGCCGCGATCGAGCGAGCCGAAGCCGAGGGCACGCTTCGTCGTGGGTCAG CAGCTGAGCTACGCGCTGCGCTCGATGAGACCACTGCCGGATTACGCGCT CGGCCGAAGCGAGCATCGGCG 624 bp (SEQ ID NO: 62) Amino acid sequence: LCAKPYLIDTIAHMAIWDRLVEVAAEQHGYVTTRDARDIGVDPVQLRLLA GRGRLERVGRGVYRVPVLPRGEHDDLAAAVSWTLGRGVISHESALALHAL ADVNPSRIHLTVPRNNHPRAAGGELYRVHRRDLQAAHVTSVDGIPVTTVA RTIKDCVKTGTDPYQLRAAIERAEAEGTLRRGSAAELRAALDETTAGLRA RPKRASA (SEQ ID NO: 81) Rv2882c RIBOSOME RECYCLING FACTOR FRR (RIBOSOME RELEASING FACTOR) (RRF) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGATTGATGAGGCT (SEQ ID NO: 42) 3' primer: TGATGATGAGAACCCCCCCCGACCTCCAGCAGCTCGCCTT (SEQ ID NO: 43) Polynucleotide sequence: ATGATTGATGAGGCTCTCTTCGACGCCGAAGAGAAAATGGAGAAGGCTGT GGCGGTGGCACGTGACGACCTGTCAACTATCCGTACCGGCCGCGCCAACC CTGGCATGTTCTCTCGGATCACCATCGACTACTACGGTGCGGCCACCCCG ATCACGCAACTGGCCAGCATCAATGTCCCCGAGGCGCGGCTAGTCGTGAT AAAGCCGTATGAAGCCAATCAGTTGCGCGCTATCGAGACTGCAATTCGCA ACTCCGACCTTGGAGTGAATCCCACCAACGACGGCGCCCTTATTCGCGTG GCCGTACCGCAGCTCACCGAAGAACGTCGGCGAGAGCTGGTCAAACAGGC AAAGCATAAGGGGGAGGAGGCCAAGGTTTCGGTGCGTAATATCCGTCGCA AAGCGATGGAGGAACTCCATCGCATCCGTAAGGAAGGCGAGGCCGGCGAG GATGAGGTCGGTCGCGCAGAAAAGGATCTCGACAAGACCACGCACCATAC GTCACCCAAATTGATGAGCTGGTTAAACACAAAGAAGGCGAGCTGCTGGA GGTC 558 bp (SEQ ID NO: 63) Amino acid sequence: MIDEALFDAEEKMEKAVAVARDDLSTIRTGRANPGMFSRITIDYYGAATP ITQLASINVPEARLVVIKPYEANQLRAIETAIRNSDLGVNPTNDGALIRV AVPQLTEERRRELVKQAKHKGEEAKVSVRNIRRKAMEELHRIRKEGEAGE DEVGRAEKDLDKTTHQYVTQIDELVKHKEGELLEV (SEQ ID NO: 82) Rv3733c CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGCCCAAGCTCAGC (SEQ ID NO.: 44) 3' primer: TGATGATGAGAACCCCCCCCGCGAGGCAGGGATTCTGGTC (SEQ ID NO: 45) Polynucleotide sequence: ATGCCCAAGCTCAGCGCGGGTGTGCTGCTGTATCGGGCGCGCGCCGGTGT CGTCGACGTCCTTCTGGCGCATCCGGGCGGCCCGTTTTGGGCGGGAAAGG ACGACGGCGCTTGGTCGATCCCGAAGGGCGAATACACCGGCGGCGAAGAT CCGTGGCTGGCCGCCCGGCGCGAGTTCTCCGAGGAGATCGGGTTGTGCGT GCCTGACGGGCCGCGAATCGACTTCGGGTCGCTGAAACAGTCCGGCGGCA AGGTGGTGACCGTGTTCGGTGTCCGGGCGGATCTGGACATCACCGACGCA CGAAGCAGCACCTTCGAATTGGACTGGCCGAAGGGCTCGGGCAAGATGCG TAAGTTCCCCGAGGTCGACCGGGTGAGCTGGTTTCCGGTAGCGCGGGCAC GCACCAAACTGCTCAAGGGGCAGCGGGGTTTTCTCGACCGGTTGATGGCG CACCCGGCCGTGGCGGGTTTGTCTGAAGGACCAGAATCCCTGCCTCGC 501 bp (SEQ ID NO: 64) Amino acid sequence: MPKLSAGVLLYRARAGVVDVLLAHPGGPFWAGKDDGAWSIPKGEYTGGED PWLAARREFSEEIGLCVPDGPRIDFGSLKQSGGKVVTVFGVRADLDITDA RSSTFELDWPKGSGKMRKFPEVDRVSWFPVARARTKLLKGQRGFLDRLMA HPAVAGLSEGPESLPR (SEQ ID NO: 83) Rv0138 CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATGTGAGCGCTTCGGAG (SEQ ID NO: 86) 3' primer: TGATGATGAGAACCCCCCCCAGGACCTCCATGCCGGCGCA (SEQ ID NO: 87) Polynucleotide sequence: GTGAGCGCTTCGGAGTTCTCCCGTGCTGAACTCGCCGCCGCCTTCGAGAA GTTCGAGAAGACCGTGGCCCGCGCCGCCGCGACGCGCGACTGGGATTGCT GGGTGCAGCACTACACCCCCGACGTCGAATACATCGAGCACGCGGCGGGC ATCATGCGAGGCCGCCAGCGGGTACGTGCCTGGATTCAAGAAACGATGAC GACCTTCCCGGGCAGTCACATGGTGGCCTTCCCGTCGCTGTGGTCGGTGA TCGACGAGTCCACCGGGCGAATTATCTGCGAATTGGACAACCCCATGCTC GACCCCGGCGACGGCAGCGTGATCAGCGCGACGAACATTTCGATCATCAC CTATGCCGGCAATGGCCAGTGGTGCCGTCAAGAAGACATCTACAACCCGT TGCGGTTCCTGCGGGCGGCGATGAAGTGGTGTCGCAAGGCGCAGGAGTTG GGCACCCTCGACGAGGACGCGGCGCGTTGGATGCGCCGGCATGGAGGTCC T SEQ ID NO: 110) Amino acid sequence: VSASEFSRAELAAAFEKFEKTVARAAATRDWDCWVQHYTPDVEYIEHAAG IMRGRQRVRAWIQETMTTFPGSHMVAFPSLWSVIDESTGRIICELDNPML DPGDGSVISATNISIITYAGNGQWCRQEDIYNPLRFLRAAMKWCRKAQEL GTLDEDAARWMRRHGGP (SEQ ID NO: 122) Rv0740 CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGCTGCCGAAGAAC (SEQ ID NO: 88) 3' primer: TGATGATGAGAACCCCCCCCGCCCTCGGCGGCGTCTTTCG (SEQ ID NO: 89) Polynucleotide sequence: ATGCTGCCGAAGAACACCAGACCCACCTCGGAAACCGCCGAAGAGTTCTG GGACAACTCGCTGTGGTGCAGCTGGGGCGACCGAGAAACGGGATACACCC GCACCGTCACGGTTTCGATCTGCCAGGTGGCGGACGGCGAACGTGAGGCC GAAGGGGTTCGGGACATGATGCGGCTGGAGTGTCCGGCTGGGCTGGATCT ACGGACACCCAACCCGGAGGCATACGAGATTACCGGTCAGCGGCCCGGAG AATTCGTGTTCGTGCTCGGCTATCTGGGGCATGTGCGGGCCATCGTGGGC AACTGTTACATCGAGATCATGCCGATGGGCACCAGGGTCGAGCTGAGCAA GTTGGCCGATGTGGCATTGGATATCGGCCGCAGTGTCGGATGCTCGGCCT ACGAGAACGACTTCACGCTGCCGGACATTCCAACGCAGTGGCGCAACCAG CCGCTGGGCTGGTACACGCAAGGCCTTGCCCCCTACCTGCCGGGGCTGTC GGACCCGAAAGACGCCGCCGAGGGC SEQ ID NO: 111) Amino acid sequence: MLPKNTRPTSETAEEFWDNSLWCSWGDRETGYTRTVTVSICQVADGEREA EGVRDMMRLECPAGLDLRTPNPEAYEITGQRPGEFVFVLGYLGHVRAIVG NCYIEIMPMGTRVELSKLADVALDIGRSVGCSAYENDFTLPDIPTQWRNQ PLGWYTQGLAPYLPGLSDPKDAAEG (SEQ ID NO: 123) Rv0733 PROBABLE ADENYLATE KINASE ADK (ATP-AMP TRANSPHOSPHORYLASE) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATGTGAGAGTTTTGTTG (SEQ ID NO: 90) 3' primer: TGATGATGAGAACCCCCCCCCTTTCCCAGAGCCCGCAACG (SEQ ID NO: 91) Polynucleotide sequence: GTGAGAGTTTTGTTGCTGGGACCGCCCGGGGCGGGCAAGGGGACGCAGGC GGTGAAGCTGGCCGAGAAGCTCGGGATCCCGCAGATCTCCACCGGCGAAC TCTTCCGGCGCAACATCGAAGAGGGCACCAAGCTCGGCGTGGAAGCCAAA CGCTACTTGGATGCCGGTGACTTGGTGCCGTCCGACTTGACCAATGAACT CGTCGACGACCGGCTGAACAATCCGGACGCGGCCAACGGATTCATCTTGG ATGGCTATCCACGCTCGGTCGAGCAGGCCAAGGCGCTTCACGAGATGCTC GAACGCCGGGGGACCGACATCGACGCGGTGCTGGAGTTTCGTGTGTCCGA GGAGGTGTTGTTGGAGCGACTCAAGGGGCGTGGCCGCGCCGACGACACCG ACGACGTCATCCTCAACCGGATGAAGGTCTACCGCGACGAGACCGCGCCG CTGCTGGAGTACTACCGCGACCAATTGAAGACCGTCGACGCCGTCGGCAC CATGGACGAGGTGTTCGCCCGTGCGTTGCGGGCTCTGGGAAAG (SEQ ID NO: 112) Amino acid sequence: VRVLLLGPPGAGKGTQAVKLAEKLGIPQISTGELFRRNIEEGTKLGVEAK RYLDAGDLVPSDLTNELVDDRLNNPDAANGFILDGYPRSVEQAKALHEML ERRGTDIDAVLEFRVSEEVLLERLKGRGRADDTDDVILNRMKVYRDETAP LLEYYRDQLKTVDAVGTMDEVFARALRALG K (SEQ ID NO: 124) Rv1065 CONSERVED HYPOTHETICAL PROTEIN 5' primer:
GAAGGAGATATACCATGCATCATCATCATCATCATGTGGTTATGCCTCTT (SEQ ID NO: 92) 3' primer: TGATGATGAGAACCCCCCCCTCCCGACCCTTCGGGCTGGT (SEQ ID NO: 93) Polynucleotide sequence: GTGGTTATGCCTCTTGTCACGCCAACCACCGCGGTTCCATCACCGGGACC CACACGGCTGCGTGTAGCCGATCTCCTGCGCGCCACCGACCAAGCCGCAG ACGACGTGCTTGGCGGGCGCTGCGACCACCTGCTACCCGACGGTGGTGTC CCGCAGACGCAGCGCTGGTACACCCGCATCCACGGTGACGAGGAGCTGGA TATCTGGCTGATTAGCTGGGTTCCCGGTCAACCGACCGAGCTGCACGACC ATGGCGGGTCCCTGGGAGCGTTGACCGTGCTGAGCGGGTCGCTCAACGAA TATCGTTGGGACGGCCGTCGGTTGCGACGGCGCCGCCTCGATGCCGGTGA TCAGGCAGGGTTCCCGTTGGGTTGGGTGCACGACGTGGTGTGGGCGCCCC GGCCGATTGGGGGGCCTGATGCGGCCGGGATGGCTGTGGCGCCAACCCTG AGCGTGCACGCCTACTCGCCGCCGCTGACGGCGATGTCGTACTACGAGAT CACCGAACGCAACACGCTGCGCCGCCAGCGCACCGAATTGACCGACCAGC CCGAAGGGTCGGGA (SEQ ID NO: 113) Amino acid sequence: VVMPLVTPTTAVPSPGPTRLRVADLLRATDQAADDVLGGRCDHLLPDGGV PQTQRWYTRIHGDEELDIWLISWVPGQPTELHDHGGSLGALTVLSGSLNE YRWDGRRLRRRRLDAGDQAGFPLGWVHDVVWAPRPIGGPDAAGMAVAPTL SVHAYSPPLTAMSYYEITERNTLRRQRTELTDQPEGSG (SEQ ID NO: 125) Rv2114 HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGTCGGCTCCCGAA (SEQ ID NO: 94) 3' primer: TGATGATGAGAACCCCCCCCGGCGGTCACCAGCGAGTAGC (SEQ ID NO: 95) Polynucleotide sequence: ATGTCGGCTCCCGAACGGGTAACCGGCTTGTCCGGGCAACGTTACGGGGA AGTCCTTCTCGTAACACCCGGGGAGGCCGGTCCACAGGCCACCGTTTACA ACAGCTTCCCGCTTAACGATTGTCCGGCCGAGCTGTGGTCCGCGCTCGAT CCGCAAGCCCTAGCCACCGAACACAAAGCGGCCACCGCCCTGCTCAACGG TCCGCGCTATTGGTTGATGAACGCCATCGAGAAGGCGCCCCAGGGCCCGC CGGTGACGAAGACCTTCGGCGGGATCGAGATGCTCCAGCAGGCCACGGTG CTGCTGTCATCGATGAACCCTGCCCCATACACCGTCAGCCAGGTCAGCCG CAACACGGTCTTTGTGTTCAACGCCGGCGAAGAGGTCTACGAACTGCAGG ACCCCAAGGGACAGCGCTGGGTGATGCAGACGTGGAGTCAAGTGGTGGAC CCCAACCTGTCCCGAGCCGACCTGCCCAAGCTGGGTGAACGGCTCAACCT GCCAGCCGGGTGGTCCTATCATACCCGCGTGCTTACCAGCGAGTTGCGGG TCGACACTACCAACCGGGAGGCCCGCGTCCTGCAAGACGACCTCACCAAC AGCTACTCGCTGGTGACCGCC (SEQ ID NO: 114) Amino acid sequence: MSAPERVTGLSGQRYGEVLLVTPGEAGPQATVYNSFPLNDCPAELWSALD PQALATEHKAATALLNGPRYWLMNAIEKAPQGPPVTKTFGGIEMLQQATV LLSSMNPAPYTVSQVSRNTVFVFNAGEEVYELQDPKGQRWVMQTWSQVVD PNLSRADLPKLGERLNLPAGWSYHTRVLTSELRVDTTNREARVLQDDLTN SYSLVTA (SEQ ID NO: 126) Rv2466c CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGCTCGAGAAGGCC (SEQ ID NO: 96) 3' primer: TGATGATGAGAACCCCCCCCGTCGAACTGAGGCGGCTCGG (SEQ ID NO: 97) Polynucleotide sequence: ATGCTCGAGAAGGCCCCCCAGAAGTCTGTCGCCGATTTCTGGTTCGATCC GCTGTGCCCGTGGTGCTGGATCACGTCGCGCTGGATCCTCGAGGTGGCAA AGGTCCGCGACATCGAGGTGAACTTCCACGTCATGAGCCTGGCAATACTC AACGAAAACCGTGACGACCTGCCCGAGCAATACCGCGAAGGCATGGCGAG GGCATGGGGACCGGTACGGGTGGCGATCGCCGCCGAGCAGGCCCATGGGG CGAAAGTCCTGGACCCGCTGTACACCGCGATGGGCAACCGGATTCACAAC CAGGGCAACCACGAACTCGACGAGGTCATCACCCAGTCGCTGGCGGACGC CGGTCTGCCCGCGGAGTTGGCCAAGGCCGCTACCAGCGACGCTTACGACA ACGCCCTGCGCAAAAGCCACCACGCCGGGATGGACGCGGTGGGCGAGGAC GTCGGTACGCCGACGATCCATGTCAATGGTGTGGCGTTCTTCGGGCCGGT GCTCTCGAAGATTCCGCGCGGCGAGGAAGCCGGCAAGCTCTGGGATGCCT CGGTTACCTTCGCTTCCTACCCGCACTTTTTTGAGCTCAAGCGGACCCGC ACCGAGCCGCCTCAGTTCGAC (SEQ ID NO: 115) Amino acid sequence: MLEKAPQKSVADFWFDPLCPWCWITSRWILEVAKVRDIEVNFHVMSLAIL NENRDDLPEQYREGMARAWGPVRVAIAAEQAHGAKVLDPLYTAMGNRIHN QGNHELDEVITQSLADAGLPAELAKAATSDAYDNALRKSHHAGMDAVGED VGTPTIHVNGVAFFGPVLSKIPRGEEAGKLWDASVTFASYPHFFELKRTR TEPPQFD (SEQ ID NO: 127) Rv0158 PROBABLE TRANSCRIPTIONAL REGULATORY PROTEIN (POSSIBLY TETR-FAMILY) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGCCATCCGACACC (SEQ ID NO: 98) 3' primer: TGATGATGAGAACCCCCCCCCGTTTCCTTCCGAGTTCCAA (SEQ ID NO: 99) Polynucleotide sequence: ATGCCATCCGACACCAGCCCCAACGGGCTAAGCCGCCGTGAGGAGTTGCT GGCTGTTGCCACCAAACTATTCGCGGCGCGCGGTTATCACGGCACCCGGA TGGACGACGTCGCCGATGTGATCGGGCTCAACAAAGCAACGGTCTATCAC TACTACGCCAGCAAGTCGCTGATCCTGTTCGACATTTACCGTCAGGCGGC CGAGGGCACCCTGGCCGCCGTGCACGACGATCCGTCCTGGACGGCCCGTG AAGCGCTGTACCAGTACACGGTCCGGCTGCTCACTGCGATCGCGAGCAAC CCCGAGCGGGCCGCCGTGTACTTCCAGGAGCAGCCCTACATCACCGAGTG GTTCACCAGCGAGCAGGTCGCCGAGGTCCGCGAGAAGGAGCAGCAAGTCT ACGAGCACGTACACGGCCTGATCGACCGCGGGATTGCCAGCGGCGAGTTC TATGAGTGCGACTCGCATGTGGTGGCGCTGGGGTACATCGGGATGACGCT GGGCAGCTACCGCTGGCTGCGGCCGAGCGGGCGCCGAACGGCCAAGGAGA TCGCGGCGGAGTTCAGCACGGCACTGCTGCGCGGGCTGATCCGCGACGAA TCGATCCGCAACCAGTCTCCGCTTGGAACTCGGAAGGAAACG (SEQ ID NO: 116) Amino acid sequence: MPSDTSPNGLSRREELLAVATKLFAARGYHGTRMDDVADVIGLNKATVYH YYASKSLILFDIYRQAAEGTLAAVHDDPSWTAREALYQYTVRLLTAIASN PEPAAVYFQEQPYITEWFTSEQVAEVREKEQQVYEHVHGLIDRGIASGEF YECDSHVVALGYIGMTLGSYRWLRPSGRRTAKEIAAEFSTALLRGLIRDE SIRNQSPLGTRKET (SEQ ID NO: 128) Rv3676 PROBABLE TRANSCRIPTIONAL REGULATORY PROTEIN (PROBABLY CRP/FNR-FAMILY) 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATGTGGACGAGATCCTG (SEQ ID NO: 100) 3' primer: TGATGATGAGAACCCCCCCCCCTCGCTCGGCGGGCCAGTC (SEQ ID NO: 101) Polynucleotide sequence: GTGGACGAGATCCTGGCCAGGGCAGGAATCTTCCAAGGCGTGGAGCCCAG CGCAATCGCCGCACTGACGAAACAGCTGCAGCCCGTCGACTTCCCCCGTG GACACACGGTCTTCGCGGAAGGGGAGCCGGGCGATCGGCTGTACATCATC ATCTCGGGGAAGGTCAAGATCGGTCGCCGGGCACCAGACGGCCGAGAAAA CCTGTTAACCATCATGGGCCCGTCGGACATGTTCGGCGAGTTGTCGATCT TCGACCCGGGTCCGCGCACGTCCAGCGCGACCACGATCACCGAGGTGCGG GCGGTGTCGATGGACCGCGACGCGCTGCGGTCATGGATCGCCGATCGTCC CGAAATCTCCGAACAGCTGCTGCGGGTGCTGGCCCGCCGGCTGCGCCGCA CCAACAACAACCTGGCCGACCTCATCTTCACCGATGTGCCCGGTCGGGTG GCCAAGCAGCTGTTGCAGCTCGCCCAGCGTTTCGGCACCCAGGAAGGTGG CGCATTGCGGGTCACCCACGACCTGACACAGGAAGAAATCGCCCAGCTGG TCGGGGCCTCACGCGAGACGGTGAACAAGGCACTGGCTGATTTCGCTCAC CGCGGCTGGATCCGCCTTGAGGGCAAGAGTGTGCTGATCTCTGACTCCGA AAGACTGGCCCGCCGAGCGAGG (SEQ ID NO: 117) Amino acid sequence: VDEILARAGIFQGVEPSAIAALTKQLQPVDFPRGHTVFAEGEPGDRLYII ISGKVKIGRRAPDGRENLLTIMGPSDMFGELSIFDPGPRTSSATTITEVR AVSMDRDALRSWIADRPEISEQLLRVLARRLRRTNNNLADLIFTDVPGRV AKQLLQLAQRFGTQEGGALRVTHDLTQEEIAQLVGASRETVNKALADFAH RGWIRLEGKSVLISDSERLARRAR (SEQ ID NO: 129) Rv2821c CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGACTACGAGCTAC (SEQ ID NO: 102) 3' primer: TGATGATGAGAACCCCCCCCAACAGCCGCGAGTTCATGGT (SEQ ID NO: 103) Polynucleotide sequence: ATGACTACGAGCTACGCCAAGATCGAGATAACCGGGACACTGACCGTCCT GACGGGCCTGCAGATCGGGGCCGGCGATGGCTTCTCCGCCATCGGCGCGG TCGACAAGCCTGTCGTTCGTGATCCGCTGAGCAGGCTGCCGATGATTCCG GGTACCAGCCTGAAGGGCAAGGTCCGCACCTTGCTGTCCCGCCAATACGG CGCCGACACAGAAACGTTTTACAGGAAGCCGAATGAGGACCACGCCCATA TCCGTCGGCTTTTCGGCGACACCGAGGAGTACATGACGGGCCGACTCGTC TTCCGCGACACGAAGCTCACCAACAAAGACGACCTCGAAGCCCGCGGCGC TAAGACTCTCACCGAGGTGAAATTCGAGAACGCCATCAACCGGGTGACCG CAAAGGCAAACCTTCGCCAGATGGAACGCGTGATCCCCGGCAGCGAGTTC GCGTTCTCACTTGTCTACGAGGTCTCCTTCGGCACCCCCGGCGAGGAACA GAAGGCGTCTCTGCCTTCCTCCGATGAGATCATCGAGGACTTCAACGCCA TCGCGCGCGGCCTGAAGTTGCTCGAACTCGACTACCTCGGCGGCAGCGGA ACCCGTGGCTACGGGCAGGTCAAGTTCAGCAACCTGAAAGCCCGCGCCGC AGTCGGCGCCCTCGACGGTTCTCTGCTGGAGAAGCTAAACCATGAACTCG CGGCTGTT (SEQ ID NO: 118) Amino acid sequence: MTTSYAKIEITGTLTVLTGLQIGAGDGFSAIGAVDKPVVRDPLSRLPMIP GTSLKGKVRTLLSRQYGADTETFYRKPNEDHAHIRRLFGDTEEYMTGRLV FRDTKLTNKDDLEARGAKTLTEVKFENAINRVTAKANLRQMERVIPGSEF AFSLVYEVSFGTPGEEQKASLPSSDEIIEDFNAIARGLKLLELDYLGGSG TRGYGQVKFSNLKARAAVGALDGSLLEKLNHELAAV (SEQ ID NO: 130) Rv1056 CONSERVED HYPOTHETICAL PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGAGCGTGGATTAC (SEQ ID NO: 104) 3' primer: TGATGATGAGAACCCCCCCCGCTGAACTGAGTGTGCGGCC (SEQ ID NO: 105) Polynucleotide sequence: ATGAGCGTGGATTACCCCCAAATGGCTGCTACCCGGGGAAGAATAGAACC GGCCCCGCGGCGAGTTCGCGGCTATCTCGGACATGTGCTCGTCTTCGACA CCAGTGCGGCGCGCTATGTCTGGGAGGTTCCCTACTACCCGCAGTACTAC ATCCCGCTGGCGGATGTCCGCATGGAGTTCCTGCGCGACGAGAACCACCC GCAGCGAGTGCAGCTGGGTCCGTCGCGGCTGCACTCCTTGGTAAGCGCCG GTCAGACCCACCGATCGGCGGCGCGGGTATTCGATGTCGACGGCGACAGC CCGGTGGCGGGCACCGTGCGTTTCAACTGGGATCCGCTGCGGTGGTTCGA GGAGGACGAGCCGATCTACGGCCATCCGCGCAATCCCTATCAGCGGGCCG ATGCGCTGCGCTCGCACCGACACGTCCGTGTCGAGCTGGACGGCATTGTG CTCGCTGACACCCGATCGCCCGTTCTGCTATTCGAAACTGGGATACCCAC AAGGTATTACATCGATCCGGCCGACATCGCTTTCGAGCATCTGGAGCCCA CCTCGACGCAGACGTTGTGTCCGTACAAGGGGACGACGTCGGGCTATTGG TCTGTGCGCGTCGGCGACGCCGTGCACCGCGACCTGGCCTGGACGTATCA CTATCCACTGCCCGCCGTTGCCCCGATCGCCGGCCTGGTGGCGTTTTACA ACGAGAAGGTCGACCTCACCGTCGACGGCGTCGCCCTGCCGCGGCCGCAC ACTCAGTTCAGC (SEQ ID NO: 119) Amino acid sequence: MSVDYPQMAATRGRIEPAPRRVRGYLGHVLVFDTSAARYVWEVPYYPQYY IPLADVRMEFLRDENHPQRVQLGPSRLHSLVSAGQTHRSAARVFDVDGDS PVAGTVRFNWDPLRWFEEDEPIYGHPRNPYQRADALRSHRHVRVELDGIV LADTRSPVLLFETGIPTRYYIDPADIAFEHLEPTSTQTLCPYKGTTSGYW SVRVGDAVHRDLAWTYHYPLPAVAPIAGLVAFYNEKVDLTVDGVALPRPH TQFS (SEQ ID NO: 131) Rv1353c PROBABLE TRANSCRIPTIONAL REGULATORY PROTEIN 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGCAGACAACCCCA (SEQ ID NO: 106) 3' primer: TGATGATGAGAACCCCCCCCACGCGCCACCGCTTTGGCCC (SEQ ID NO: 107) Polynucleotide sequence: ATGCAGACAACCCCAGGCAAGCGTCAACGACGGCAGCGCGGATCCATCAA CCCCGAGGACATCATCAGCGGCGCATTCGAACTCGCCCAGCAGGTATCGA TAGACAACTTGAGCATGCCATTGCTCGGCAAACACCTTGGCGTCGGGGTC ACCAGCATCTACTGGTACTTCCGCAAGAAGGACGATCTGCTCAACGCGAT GACCGACCGCGCTTTGAGCAAGTACGTGTTCGCTACCCCGTACATCGAAG
CCGGCGACTGGCGCGAAACGTTGCGCAATCATGCCCGCTCGATGCGGAAG ACGTTCGCGGACAACCCCGTACTGTGCGATCTGATACTGATTCGAGCGGC GCTGTCCCCGAAAACGGCGCGGTTGGGCGCCCAAGAGATGGAGAAGGCCA TCGCCAATCTGGTGACGGCGGGCCTGTCGCTCGAAGACGCTTTCGACATC TACTCGGCGGTTTCGGTCCACGTGCGCGGATCGGTGGTGCTAGATCGGCT CTCCCGCAAGAGCCAGTCGGCGGGCAGCGGACCATCCGCCATTGAACACC CCGTGGCCATCGATCCCGCGACGACTCCGCTGCTTGCTCACGCAACTGGG AGGGGGCATCGGATCGGGGCCCCCGATGAAACCAATTTCGAATATGGTCT CGAATGCATCCTCGACCATGCTGGCCGGTTGATCGAACAAAGCTCGAAAG CCGCTGGTGAGGTCGCAGTGCGCCGCCCCACGGCCACCGCCGATGCGCCT ACGCCGGGCGCGCGGGCCAAAGCGGTGGCGCGT (SEQ ID NO: 120) Amino acid sequence: MQTTPGKRQRRQRGSINPEDIISGAFELAQQVSIDNLSMPLLGKHLGVGV TSIYWYFRKKDDLLNAMTDRALSKYVFATPYIEAGDWRETLRNHARSMRK TFADNPVLCDLILIRAALSPKTARLGAQEMEKAIANLVTAGLSLEDAFDI YSAVSVHVRGSVVLDRLSRKSQSAGSGPSAIEHPVAIDPATTPLLAHATG RGHRIGAPDETNFEYGLECILDHAGRLIEQSSKAAGEVAVRRPTATADAP TPGARAKAVAR (SEQ ID NO: 132) Rv2528c PROBABLE RESTRICTION SYSTEM PROTEIN MRR 5' primer: GAAGGAGATATACCATGCATCATCATCATCATCATATGACGATCCCTGAT (SEQ ID NO: 108) 3' primer: TGATGATGAGAACCCCCCCCCAGGCCATCAAAAAAGTCCT (SEQ ID NO: 109) Polynucleotide sequence: ATGACGATCCCTGATGCCCAGACGTTGATGCGGCCGATTCTCGCGTATCT TGCCGATGGACAAGCGAAGTCGGCCAAGGACGTCATCGCGGCGATGTCCG ACGAGTTCGGTCTGTCCGACGACGAGCGGGCGCAGATGTTGCCCAGCGGT CGGCAAAGGACCATGTACGACAGGGTGCACTGGTCTCTCACTCACATGTC GCAGGCCGGATTGCTCGACCGTCCCACGCGGGGCCACGTCCAGGTCACGG ACACGGGCCGTCAAGTCCTGAAGGCGCATCCCGAGCGCGTCGACATGGCT GTGCTGCGGGAGTTCCCGTCGTACATCGCTTTTCGTGAGCGAACCAAAGC CAAGCAGCCAGTCGACGCGACCGCCAAGCGACCGTCCGGGGACGATGTGC AGGTCTCACCCGAGGATCTCATCGACGCTGCGCTTGCGGAGAACCGGGCA GCCGTCGAGGGGGAGATCCTGAAGAAGGCACTCACGTTGTCGCCCACCGG GTTTGAAGATCTGGTTATCAGACTTTTGGAGGCGATGGGTTACGGGCGAG CCGGCGCGGTGGAACGGACGAGTGCCTCCGGTGACGCTGGCATCGACGGA ATCATCAGCCAGGACCCGCTCGGGCTGGACCGCATCTACGTGCAGGCCAA GCGATACGCCGTCGACCAAACGATTGGCCGGCCGAAGATCCACGAGTTCG CCGGCGCCCTCCTGGGCAAGCAGGGCGACCGGGGCGTCTACATCACCACG TCATCGTTTTCCCGCGGTGCCCGCGAGGAAGCTGAGCGGATCAACGCCCG GATCGAACTCATCGACGGCGCTCGGCTGGCCGAGCTGCTCGTGCGGTATC GAGTCGGTGTCCAGGCGGTGCAGACCGTCGAACTCTTACGGCTCGACGAG GACTTTTTTGATGGCCTG (SEQ ID NO: 121) Amino acid sequence: MTIPDAQTLMRPILAYLADGQAKSAKDVIAAMSDEFGLSDDERAQMLPSG RQRTMYDRVHWSLTHMSQAGLLDRPTRGHVQVTDTGRQVLKAHPERVDMA VLREFPSYIAFRERTKAKQPVDATAKRPSGDDVQVSPEDLIDAALAENRA AVEGEILKKALTLSPTGFEDLVIRLLEAMGYGRAGAVERTSASGDAGIDG IISQDPLGLDRIYVQAKRYAVDQTIGRPKIHEFAGALLGKQGDRGVYITT SSFSRGAREEAERINARIELIDGARLAELLVRYRVGVQAVQTVELLRLDE DFFDGL (SEQ ID NO: 133)
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:
Promoter Fragment: (SEQ ID NO:6)
TABLE-US-00002 5'CGGTCACGCTTGGGACTGCCATAGGCTGGCCCGGTGATGCCGGCCACG ATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGA CTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTA ACTTTAAGAAGGAGATATACC 3'
Terminator Fragment: TABLE-US-00003 (SEQ ID NO: 7)
TABLE-US-00003 5'GGGGGGGGTTCTCATCATCATCATCATCATTAATAAAAGGGCGAATTC CAGCACACTGGCGGCCGTTACTAGTGGATCCGGCTGCTAACAAAGCCCGA AAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACC CCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAA CTATATCCGGAGCGACTCCCACGGCACGTTGGCAAGCTCG 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˜1.0μg PCR product was used as template, producing approximately 0.5˜5.0μg of protein per template.
MagneHis nickel-coated magnetic beads (Promega) were used to purify the expressed proteins. 15μl of Ni-magnetic beads (Promega) were pipetted into each well of a microtiter plate. To each well 50 μl wash buffer (50 mM NaHPO4, pH 8.0, 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 150μl 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 rabbit anti-Mtb serum. The blots were washed and then incubated with alkaline phosphatase-conjugated goat-anti rabbit serum secondary antibody. Colorimetric development was used to develop the blots. The results of these analyses are shown in FIG. 6.
The wells of Nunc-Immuno MaxiSorp 96-well plates were coated with 5 μl of expressed protein diluted in 95 μl PBS. The plates were mixed well on a shaker and then incubated overnight at 4° C. The plates were washed with PBS+0.05% Tween-20 for 5 min. with shaking at 200 rpm. 200 μl of PBS+1% BSA blocking solution was added and the plates incubated for 1 hr at room temperature, with shaking at 150 rpm. The blocking buffer was removed and 100 μl primary antibody (04.E293.1.11.WCL(-)LAM rabbit polyclonal antibodies 1:200000 diluted in blocking solution) was added. Following 1 hr incubation at room temperature with shaking at 150 rpm, the plates were washed 3 times with PBS+0.05% Tween-20. 100 μl second antibody (Anti-Rabbit IgG(H+L)-HRP conjugated, (Promega) diluted 1:2500 in blocking solution was added to each well and the plates were incubated for 1 hr at room temperature, with shaking at 150 rpm. After washing 3 times, 100 μl TMB substrate solution (Promega) was added to each well and the blue color was allowed to develop for 15 min at room temperature without shaking. 100 μl of 1N HCl was added to each well to stop the reaction and change the blue color to yellow. The plates were read in a spectrophotometer at 450 nm after 30 min. The results of this analysis are shown in FIG. 6.
As shown in FIG. 6, rabbit anti-Mtb serum identified 19 and 12 proteins that were reactive to the anti-serum Western blot and ELISA, respectively. The results showed a strong correlation in `hits` between the two methods. In addition, a few antigen proteins at low abundance exhibited high reactivity relative to the others, suggesting the presence of strong B-cell epitopes, thus making them premier candidates for additional study.
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-γ purified MAb (Clone 1-DIK) (MABTECH #3420-3) (Mabtech, Naka, Sweden)
Anti-EFN-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 (0.2μM 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-DIK) 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 dH2O 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.
Cellular Vaccine Antigen Screen
A human volunteer was immunized with irradiated sporozoites from P. falciparum, the infectious agent responsible for malaria. Dendritic cells from the volunteer were isolated and cultured. Recombinant CSP polypeptide from P. falciparum was delivered to dendritic cells with or without polypeptide delivery reagents described in U.S. patent application Ser. No. 09/738,046, entitled "Intracellular Protein Delivery Reagent," which is hereby incorporated by reference in its entirety. T-cells isolated from the immunized volunteer were added to the cultures. The EliSpotassay identified 120 CSP antigen specific T-cells out of 250,000 T-cells that were added to the culture when CSP was added to the culture together with said delivery reagents. When CSP was added without said delivery reagents, the signal was barely above background.
DNA Immunization of Mice
Experiments were set up with five animals per group, consisting of four week old BALB/c female mice, averaging 40 animals per experiment. These mice were immunized IM in each tibialis anterior muscle with 50 μg plasmid DNA or transcriptionally active PCR fragment encoding selected Mtb antigens, 3 times at 3 week intervals.
Sera was collected 10 days after each immunization for antibody studies. Blood samples (˜50 ul) were collected from the mice by orbital bleed with a sterilized pasture pipette. The mice were bled about once a week at a volume of approximately 50 μl.
Splenocytes were harvested at 14 days after the 3rd immunization and pooled for T-cell studies such as IFN-γ ELIspot assays. Tissue collections were performed on animals euthanized via CO2 (SOP 98.19) at the end of the experiment. The experiments can be five animals/group, averaging 40 animals/experiment×4 experiments for a total of 160 mice.
Preparation of Human Dendritic Cells
Dendritic cells were ordered from Allcells: Cat # PB002 (NPB-Mononuclear Cells). The cells were in 50 mL buffer. The cells were counted immediately, the total number was 312.5×106. The cells were pelleted, and resuspended in 25 mL RPMI-1640 containing DNAse. This solution (30 μg/mL) was incubated for 5 minutes at room temperature. The cells were washed twice with complete medium. The cells were resuspended at 10×106 cells/3 mL. Twelve 10 mm dishes containing 10 mL complete medium in each dish were used. The cells were incubated at 37° C. for 3 hours. The non-adherent cells were removed by gently shaking plates and aspirating the supernatant. Afterwards, the dishes containing adherent cells were washed 3 times with 10 mL of RPMI-1640 containing 2% Human Serum. 10 mL of culture medium were added to each plate containing 50 ng/mL GM-CSF and 500 u/mL IL-4. This culture medium was added until day 4. After day 4, culture medium without GM-CSF and IL-4 was added. The transfection was done on day 5. The complete medium consisted of RPMI-1640 (455 mL), 5% Human AB Serum (25 mL), Non-essential Amino Acids (5 mL), Sodium Pyruvate (5 mL), L-Glutamine (5 mL), and Penicillin-Streptomycin (5 mL).
Generation of Dendritic Cells from Mouse Bone Marrow
Cells were taken from the bones of one mouse (2 femur and 2 tibiae without removing the macrophages). The red blood cells were obtained from the bone marrow and lysed. The cells were counted (51×106 cells, total) and cultured in a growth medium (2.5×106 cells/plate, 10 mL/plate) for 8 days before transfection. On day 4 another 10 mL of growth medium was added. On day 6, 10 mL of the old medium was taken from each plate and the cells were pelleted. The cells were resuspended in 10 mL medium with 10 ng/mL GM-CSF and 2.5 ng/mL IL-4. The cells were placed back into the culture. The cells were cultured until transfection on day 8. On the day of transfection, 2.5×106 cells were harvested from each dish. The growth medium for mbmDC contained DMEM/Iscove, 10% FCS, 50 uM β-mercaptoethanol, 1× Penicillin/Streptomycin, 2 mM L-Glutamine, 10 mM Hepes, 1× Non-essential amino acids, 20 ng/mL rmGM-CSF, and 5 ng/mL rmIL-4.
Adding an HA Epitope Tag
Oligos were designed using TAP promoter and terminator fragments from pCMVm and pTP-SV40, respectively, and adding the nucleotide sequence encoding the HA epitope tag. For adding the HA epitope to the 5' end of the coding sequence the following sequences is used: TABLE-US-00004 Promoter 5': CCGCCATGTTGACATTG (SEQ ID NO: 2) Promoter 3': GGCAGATCTGGGAGGCTAGCGTAATCCGGAACATCG (SEQ ID NO: 3) TATGGGTACATTGTTAAGTCGACGGTGC
For adding the HA epitope to the 3' end of the coding sequence, the following sequences is used:
TABLE-US-00004 TABLE-US-00005 Terminator 5': GATCCCGGGTACCCATACGATGTTCCGGATTACGCT (SEQ ID NO: 4) TAGGGGAGATCTCAGACATG Terminator 3': CAGGATATCATGCCTGCAGGACGACTCTAGAG (SEQ ID NO: 5)
The method includes:
PCR is used to amplify a new HA-promoter utilizing pCMVm as a template and a new HA-terminator utilizing pTP-SV40 as a template. The resulting PCR products are gel purified using QIAGEN QIAquick Gel Extraction Kit (Qiagen, Seattle, Wash.). The PCR products and both plasmids (pCMVm & pTP-SV40) are digested with EcoRV and BglII restriction enzymes. All digested products are gel purified using QIAquick Gel Extraction Kit. The HA-promoter and HA-terminator are ligated separately into the digested pCMVm and pTP-SV40 plasmids. These plasmids are transformed into DH5, grown overnight on LB plates containing Kanamycin, colonies are selected and grown in LB media containing Kanamycin. QIAGEN QIAprep Spin Miniprep Kit is used to isolate plasmids. Plasmids are digested using EcoRV and BglII Digests are run on a gel to identify clones containing plasmid with insert of correct size. The plasmids are sequenced to confirm inserts are correct. A prep culture is grown, plasmids are isolated, plasmids are digested with EcoRV and BglII, and promoter and terminator fragments are gel purified. Epi-TAP-5 HA and Epi-TAP-3'HA kits are used.
Intracellular Cytokine Staining (ICS)
Bone marrow derived dendritic cells (BMDCs) were prepared by culturing bone marrow cell suspensions with RPMI tissue culture media plus 10% fetal bovine serum and GM-CSF (20 ng/ml) for 6-7 days at 37° C., 5% CO2. Cells were then primed with 1μg/ml of antigen for 4 hrs at 37° C., 5% CO2.
Cell suspensions obtained from naive or M. tuberculosis infected mice were used as a source of CD4 T cells. CD4 T cells are isolated by magnetic cell sorting and overlaid onto BMDC primed with specific antigens and cultured at 37° C. for 24 hrs. After this time T cells were harvested and stained for CD3/CD4/intracellular IFNγ and analyzed by flow cytometry.
The sequences disclosed in Table 1 yielded positive results in at least one assay described herein, e.g. Western blot, ELISA or ICS.
In One Embodiment the Method Includes Detection of Antigen-Specific CD4.sup.+ T-Cell Responses by Intracellular Cytokine Staining (ICS).
A panel of immunogenic Mtb proteins discovered in Phase I studies that were recognized by rabbit anti-TB sera was selected for further analysis to determine if these proteins could lead to enhanced induction of CD4.sup.+ T-cells. Thirty-six purified Mtb proteins along with positive controls, culture filtrate proteins (CFP), and recombinant ESAT-6, were included in the ICS assay. The results are summarized in Table 2 and demonstrate that 11 of the 36 proteins significantly stimulated CD4.sup.+ T-cell responses. Moreover, with equal protein amounts used, 6 Mtb proteins showed greater stimulatory activity than that of ESAT-6. TABLE-US-00006 TABLE 2 Antigen-specific stimulation of CD4.sup.+ T-cells ID Rv3733c Rv0138 Rv0740 Rv0733 Rv0009 Rv2882c Rv1065 Rv2613c Rv0475 Rv2114 Rv2466c Rv3763 Rv2031c % T-cells 4.3 4.3 8.3 3.7 4.0 3.6 2.8 2.0 2.1 2.9 2.6 2.5 2.2 ID Rv1347c Rv0158Rv3676 Rv2821Rv2108 Rv3226c Rv1056 Rv0815c Rv3117 Rv1073 Rv0097 ESAT-6 Media % T-cells 2.0 2.1 1.5 1.5 1.3 2.5 2.2 1.7 1.9 1.6 1.4 3.6 1.4
One μg each from 36 purified Mtb proteins, along with the control protein ESAT-6, were incubated with mouse dendritic cells for 24 hr. Spleen cells harvested from Mtb-infected mice were added and incubated for an additional 72 hr. The splenocytes were labeled with cychrome-conjugated anti-CD4 antibody and then stained with fluorescein-conjugated anti-.γIFNγhe cells were washed, fixed and analyzed by flow cytometry. The "% T-cells" indicates the percentage of CD4.sup.+ T-cells that released γIFN. Based on previous studies, the percent value at or above 2.5% is significant.
133169DNAHomo sapiens 1atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60tcgcccagc 69217DNAArtificial SequenceSynthetic Oligonucleotide Primer 2ccgccatgtt gacattg 17364DNAArtificial SequenceSynthetic Oligonucleotide Primer 3ggcagatctg ggaggctagc gtaatccgga acatcgtatg ggtacattgt taagtcgacg 60gtgc 64456DNAArtificial SequenceSynthetic Oligonucleotide Primer 4gatcccgggt acccatacga tgttccggat tacgcttagg ggagatctca gacatg 56532DNAArtificial SequenceSynthetic Oligonucleotide Primer 5caggatatca tgcctgcagg acgactctag ag 326169DNAArtificial SequenceSynthetic Oligonucleotide Primer 6cggtcacgct tgggactgcc ataggctggc ccggtgatgc cggccacgat gcgtccggcg 60tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac 120ggtttccctc tagaaataat tttgtttaac tttaagaagg agatatacc 1697238DNAArtificial SequenceSynthetic Oligonucleotide Primer 7ggggggggtt ctcatcatca tcatcatcat taataaaagg gcgaattcca gcacactggc 60ggccgttact agtggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg 120ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt 180ttttgctgaa aggaggaact atatccggag cgactcccac ggcacgttgg caagctcg 238850DNAArtificial SequenceSynthetic Oligonucleotide Primer 8gaaggagata taccatgcat catcatcatc atcatatggc caccaccctt 50940DNAArtificial SequenceSynthetic Oligonucleotide Primer 9tgatgatgag aacccccccc gttggtggac cggatctgaa 401050DNAArtificial SequenceSynthetic Oligonucleotide Primer 10gaaggagata taccatgcat catcatcatc atcatgtgaa gcgtggactg 501140DNAArtificial SequenceSynthetic Oligonucleotide Primer 11tgatgatgag aacccccccc ggaacaggtc acctcgattt 401250DNAArtificial SequenceSynthetic Oligonucleotide Primer 12gaaggagata taccatgcat catcatcatc atcatatggc caatccgttc 501340DNAArtificial SequenceSynthetic Oligonucleotide Primer 13tgatgatgag aacccccccc ctgaccgtag ggctgctcgg 401450DNAArtificial SequenceSynthetic Oligonucleotide Primer 14gaaggagata taccatgcat catcatcatc atcatatgac gcttaaggtc 501540DNAArtificial SequenceSynthetic Oligonucleotide Primer 15tgatgatgag aacccccccc tgccgcgtat cccggcgtct 401650DNAArtificial SequenceSynthetic Oligonucleotide Primer 16gaaggagata taccatgcat catcatcatc atcatatggc tgaaaactcg 501740DNAArtificial SequenceSynthetic Oligonucleotide Primer 17tgatgatgag aacccccccc cttctgggtg accttcttgg 401850DNAArtificial SequenceSynthetic Oligonucleotide Primer 18gaaggagata taccatgcat catcatcatc atcatatggc acgctgcgat 501940DNAArtificial SequenceSynthetic Oligonucleotide Primer 19tgatgatgag aacccccccc gcttcccaac tcgatcgggg 402050DNAArtificial SequenceSynthetic Oligonucleotide Primer 20gaaggagata taccatgcat catcatcatc atcatatgac caaacccaca 502140DNAArtificial SequenceSynthetic Oligonucleotide Primer 21tgatgatgag aacccccccc cgcagccgtg gtcggagctt 402250DNAArtificial SequenceSynthetic Oligonucleotide Primer 22gaaggagata taccatgcat catcatcatc atcatatggc acgctgcgat 502340DNAArtificial SequenceSynthetic Oligonucleotide Primer 23tgatgatgag aacccccccc gcttcccaac tcgatcgggg 402450DNAArtificial SequenceSynthetic Oligonucleotide Primer 24gaaggagata taccatgcat catcatcatc atcatgtgag tgacgaggac 502540DNAArtificial SequenceSynthetic Oligonucleotide Primer 25tgatgatgag aacccccccc tggttgccga gcccactcgg 402650DNAArtificial SequenceSynthetic Oligonucleotide Primer 26gaaggagata taccatgcat catcatcatc atcatatgtg cggacggttt 502740DNAArtificial SequenceSynthetic Oligonucleotide Primer 27tgatgatgag aacccccccc cagcagctgg atctgctcgg 402850DNAArtificial SequenceSynthetic Oligonucleotide Primer 28gaaggagata taccatgcat catcatcatc atcatgtgcc agagctggag 502940DNAArtificial SequenceSynthetic Oligonucleotide Primer 29tgatgatgag aacccccccc gtccgccagc ttgaccgact 403050DNAArtificial SequenceSynthetic Oligonucleotide Primer 30gaaggagata taccatgcat catcatcatc atcatatggc agactgtgat 503140DNAArtificial SequenceSynthetic Oligonucleotide Primer 31tgatgatgag aacccccccc ggagatggtg atcgactcga 403250DNAArtificial SequenceSynthetic Oligonucleotide Primer 32gaaggagata taccatgcat catcatcatc atcatatggg ggcgcagccg 503340DNAArtificial SequenceSynthetic Oligonucleotide Primer 33tgatgatgag aacccccccc acgtcgtgat gtcaacgtgt 403450DNAArtificial SequenceSynthetic Oligonucleotide Primer 34gaaggagata taccatgcat catcatcatc atcatatgat gcaccgaacc 503540DNAArtificial SequenceSynthetic Oligonucleotide Primer 35tgatgatgag aacccccccc tcggcttcgt ggtaaacccg 403650DNAArtificial SequenceSynthetic Oligonucleotide Primer 36gaaggagata taccatgcat catcatcatc atcatatgcc caatttctgg 503740DNAArtificial SequenceSynthetic Oligonucleotide Primer 37tgatgatgag aacccccccc aaacttagga tgttccttgt 403850DNAArtificial SequenceSynthetic Oligonucleotide Primer 38gaaggagata taccatgcat catcatcatc atcatatggc cgacgctgac 503940DNAArtificial SequenceSynthetic Oligonucleotide Primer 39tgatgatgag aacccccccc gtcgcggagc acaacgactc 404050DNAArtificial SequenceSynthetic Oligonucleotide Primer 40gaaggagata taccatgcat catcatcatc atcatttgtg tgcaaaaccg 504140DNAArtificial SequenceSynthetic Oligonucleotide Primer 41tgatgatgag aacccccccc cgccgatgct cgcttcggcc 404250DNAArtificial SequenceSynthetic Oligonucleotide Primer 42gaaggagata taccatgcat catcatcatc atcatatgat tgatgaggct 504340DNAArtificial SequenceSynthetic Oligonucleotide Primer 43tgatgatgag aacccccccc gacctccagc agctcgcctt 404450DNAArtificial SequenceSynthetic Oligonucleotide Primer 44gaaggagata taccatgcat catcatcatc atcatatgcc caagctcagc 504540DNAArtificial SequenceSynthetic Oligonucleotide Primer 45tgatgatgag aacccccccc gcgaggcagg gattctggtc 4046432DNAMycobacterium tuberculosis 46atggccacca cccttcccgt tcagcgccac ccgcggtccc tcttccccga gttttctgag 60ctgttcgcgg ccttcccgtc attcgccgga ctccggccca ccttcgacac ccggttgatg 120cggctggaag acgagatgaa agaggggcgc tacgaggtac gcgcggagct tcccggggtc 180gaccccgaca aggacgtcga cattatggtc cgcgatggtc agctgaccat caaggccgag 240cgcaccgagc agaaggactt cgacggtcgc tcggaattcg cgtacggttc cttcgttcgc 300acggtgtcgc tgccggtagg tgctgacgag gacgacatta aggccaccta cgacaagggc 360attcttactg tgtcggtggc ggtttcggaa gggaagccaa ccgaaaagca cattcagatc 420cggtccacca ac 43247477DNAMycobacterium tuberculosis 47gtgaagcgtg gactgacggt cgcggtagcc ggagccgcca ttctggtcgc aggtctttcc 60ggatgttcaa gcaacaagtc gactacagga agcggtgaga ccacgaccgc ggcaggcacg 120acggcaagcc ccggcgccgc ctccgggccg aaggtcgtca tcgacggtaa ggaccagaac 180gtcaccggct ccgtggtgtg cacaaccgcg gccggcaatg tcaacatcgc gatcggcggg 240gcggcgaccg gcattgccgc cgtgctcacc gacggcaacc ctccggaggt gaagtccgtt 300gggctcggta acgtcaacgg cgtcacgctg ggatacacgt cgggcaccgg acagggtaac 360gcctcggcaa ccaaggacgg cagccactac aagatcactg ggaccgctac cggggtcgac 420atggccaacc cgatgtcacc ggtgaacaag tcgttcgaaa tcgaggtgac ctgttcc 47748810DNAMycobacterium tuberculosis 48atggccaatc cgttcgttaa agcctggaag tacctcatgg cgctgttcag ctcgaagatc 60gacgagcatg ccgaccccaa ggtgcagatt caacaggcca ttgaggaagc acagcgcacc 120caccaagcgc tgactcaaca ggcggcgcaa gtgatcggta accagcgtca attggagatg 180cgactcaacc gacagctggc ggacatcgaa aagcttcagg tcaatgtgcg ccaagccctg 240acgctggccg accaggccac cgccgccgga gacgctgcca aggccaccga atacaacaac 300gccgccgagg cgttcgcagc ccagctggtg accgccgagc agagcgtcga agacctcaag 360acgctgcatg accaggcgct tagcgccgca gctcaggcca agaaggccgt cgaacgaaat 420gcgatggtgc tgcagcagaa gatcgccgag cgaaccaagc tgctcagcca gctcgagcag 480gcgaagatgc aggagcaggt cagcgcatcg ttgcggtcga tgagtgagct cgccgcgcca 540ggcaacacgc cgagcctcga cgaggtgcgc gacaagatcg agcgtcgcta cgccaacgcg 600atcggttcgg ctgaacttgc cgagagttcg gtgcagggcc ggatgctcga ggtggagcag 660gccgggatcc agatggccgg tcattcacgg ttggaacaga tccgcgcatc gatgcgcggt 720gaagcgttgc cggccggcgg gaccacggct acccccagac cggccaccga gacttctggc 780ggggctattg ccgagcagcc ctacggtcag 81049867DNAMycobacterium tuberculosis 49atgacgctta aggtcaaagg cgagggactc ggtgcgcagg tcacaggggt cgatcccaag 60aatctggacg atataaccac cgacgagatc cgggatatcg tttacacgaa caagctcgtt 120gtgctaaaag acgtccatcc gtctccgcgg gagttcatca aactcggcag gataattgga 180caaatcgttc cgtattacga acccatgtac catcacgaag accacccgga gatctttgtc 240tcctccactg aggaaggtca gggggtccca aaaaccggcg cgttctggca tatcgactat 300atgtttatgc cggaaccttt cgcgttttcc atggtgctgc cgctggcggt gcctggacac 360gaccgcggga cctatttcat cgatctcgcc agggtctggc agtcgctgcc cgccgccaag 420cgagacccgg cccgcggaac cgtcagcacc cacgaccctc gacgccacat caagatccga 480cccagcgacg tctaccggcc catcggagag gtatgggacg agatcaaccg gaccacgccc 540ccaataaagt ggcctacggt catccggcac ccaaagaccg gccaagagat cctctacatc 600tgcgcgacgg gcaccaccaa gatcgaggac aaggacggca atccggttga tccggaggtg 660ctgcaagaac tcatggccgc gaccggacag ctcgatcctg agtaccagtc gccgttcata 720catactcagc actaccaggt tggcgacatc atcttgtggg acaaccgggt tctcatgcac 780cgagcgaagc acggcagcgc cgcgggcact ctgacgacct accgcctgac catgcttgat 840ggcctcaaga cgccgggata cgcggca 86750597DNAMycobacterium tuberculosis 50atggctgaaa actcgaacat tgatgacatc aaggctccgt tgcttgccgc gcttggagcg 60gccgacctgg ccttggccac tgtcaacgag ttgatcacga acctgcgtga gcgtgcggag 120gagactcgta cggacacccg cagccgggtc gaggagagcc gtgctcgcct gaccaagctg 180caggaagatc tgcccgagca gctcaccgag ctgcgtgaga agttcaccgc cgaggagctg 240cgtaaggccg ccgagggcta cctcgaggcc gcgactagcc ggtacaacga gctggtcgag 300cgcggtgagg ccgctctaga gcggctgcgc agccagcaga gcttcgagga agtgtcggcg 360cgcgccgaag gctacgtgga ccaggcggtg gagttgaccc aggaggcgtt gggtacggtc 420gcatcgcaga cccgcgcggt cggtgagcgt gccgccaagc tggtcggcat cgagctgcct 480aagaaggctg ctccggccaa gaaggccgct ccggccaaga aggccgctcc ggccaagaag 540gcggcggcca agaaggcgcc cgcgaagaag gcggcggcca agaaggtcac ccagaag 59751831DNAMycobacterium tuberculosis 51atggcacgct gcgatgtcct ggtctccgcc gactgggctg agagcaatct gcacgcgccg 60aaggtcgttt tcgtcgaagt ggacgaggac accagtgcat atgaccgtga ccatattgcc 120ggcgcgatca agttggactg gcgcaccgac ctgcaggatc cggtcaaacg tgacttcgtc 180gacgcccagc aattctccaa gctgctgtcc gagcgtggca tcgccaacga ggacacggtg 240atcctgtacg gcggcaacaa caattggttc gccgcctacg cgtactggta tttcaagctc 300tacggccatg agaaggtcaa gttgctcgac ggcggccgca agaagtggga gctcgacgga 360cgcccgctgt ccagcgaccc ggtcagccgg ccggtgacct cctacaccgc ctccccgccg 420gataacacga ttcgggcatt ccgcgacgag gtcctggcgg ccatcaacgt caagaacctc 480atcgacgtgc gctctcccga cgagttctcc ggcaagatcc tggcccccgc gcacctgccg 540caggaacaaa gccagcggcc cggacacatt cctggtgcca tcaacgtgcc gtggagcagg 600gccgccaacg aggacggcac cttcaagtcc gatgaggagt tggccaagct ttacgccgac 660gccggcctag acaacagcaa ggaaacgatt gcctactgcc gaatcgggga acggtcctcg 720cacacctggt tcgtgttgcg ggaattactc ggacaccaaa acgtcaagaa ctacgacggc 780agttggacag aatacggctc cctggtgggc gccccgatcg agttgggaag c 83152630DNAMycobacterium tuberculosis 52atgaccaaac ccacatccgc tggccaggcc gacgacgcgc tggttcggct agcccgcgag 60cgattcgacc tacctgacca ggtacgacgc ctcgcccgcc cgcccgttcc atcgttggag 120ccgccatacg ggttgcgggt cgcacagctg accgacgcgg agatgttggc ggagtggatg 180aaccgtcctc atctggcggc ggcctgggag tacgactggc cggcgtcacg ttggcgtcaa 240cacctgaacg cccaacttga gggaacctat tcgttgccat tgatcggcag ctggcacgga 300acagatggtg gttatctcga attatactgg gcagcaaagg atttgatttc tcactactac 360gacgcagacc cctacgattt ggggctgcac gcggccatcg cggacttgtc gaaggtcaat 420cggggcttcg gcccgctgct gctaccgcgg atcgtggcca gcgtctttgc caacgagccg 480cgttgccggc ggatcatgtt cgaccccgat caccgcaaca ccgcgacccg tcggttgtgt 540gagtgggccg gatgcaagtt cctcggtgag catgacacga caaaccggcg catggcgctc 600tacgctttgg aagctccgac cacggctgcg 63053831DNAMycobacterium tuberculosis 53atggcacgct gcgatgtcct ggtctccgcc gactgggctg agagcaatct gcacgcgccg 60aaggtcgttt tcgtcgaagt ggacgaggac accagtgcat atgaccgtga ccatattgcc 120ggcgcgatca agttggactg gcgcaccgac ctgcaggatc cggtcaaacg tgacttcgtc 180gacgcccagc aattctccaa gctgctgtcc gagcgtggca tcgccaacga ggacacggtg 240atcctgtacg gcggcaacaa caattggttc gccgcctacg cgtactggta tttcaagctc 300tacggccatg agaaggtcaa gttgctcgac ggcggccgca agaagtggga gctcgacgga 360cgcccgctgt ccagcgaccc ggtcagccgg ccggtgacct cctacaccgc ctccccgccg 420gataacacga ttcgggcatt ccgcgacgag gtcctggcgg ccatcaacgt caagaacctc 480atcgacgtgc gctctcccga cgagttctcc ggcaagatcc tggcccccgc gcacctgccg 540caggaacaaa gccagcggcc cggacacatt cctggtgcca tcaacgtgcc gtggagcagg 600gccgccaacg aggacggcac cttcaagtcc gatgaggagt tggccaagct ttacgccgac 660gccggcctag acaacagcaa ggaaacgatt gcctactgcc gaatcgggga acggtcctcg 720cacacctggt tcgtgttgcg ggaattactc ggacaccaaa acgtcaagaa ctacgacggc 780agttggacag aatacggctc cctggtgggc gccccgatcg agttgggaag c 83154585DNAMycobacterium tuberculosis 54gtgagtgacg aggaccgcac ggatcgggcc accgaggacc acaccatctt cgatcggggt 60gtcggccagc gcgaccagct gcagcggtta tggaccccct accggatgaa ctacctggcc 120gaagcgccag tgaagcgtga ccccaattcc tcggccagcc ctgcgcagcc gttcaccgag 180atcccgcagc tgtccgacga agagggtctg gtggtcgctc gtggcaagct ggtctacgcc 240gtgctcaacc tgtacccgta caaccccggg cacttgatgg tggtgcccta tcgtcgggta 300tccgaactcg aggatctcac cgatttggag agcgccgagt tgatggcgtt cacccagaag 360gcgattcgcg tgatcaagaa cgtgtcgcgt ccgcacggct tcaatgtcgg cctgaaccta 420gggacatcgg cgggcgggtc gctggccgag cacctgcacg tgcatgtggt gccacggtgg 480ggtggcgatg cgaatttcat caccatcatc gggggctcca aggtgattcc gcagctgctg 540cgcgacaccc gtcggctgct tgccaccgag tgggctcggc aacca 58555756DNAMycobacterium tuberculosis 55atgtgcggac ggtttgcggt caccactgat ccggcccagc tggccgagaa aatcacggcc 60atagacgagg ccaccgggtg cggtggcggg aagacgagct acaacgtggc acccaccgac 120acgatcgcga cagtggtgtc ccgccacagc gagcccgacg acgagcccac ccgccgggtg 180cggctcatgc gctggggact gattccgtcg tggatcaagg ccgggcccgg cggcgcaccc 240gatgccaaag gcccaccgct gatcaacgcc cgcgccgata aggtcgccac gtcgccggcg 300ttccggagtg cggtcagaag taagcgttgc ctggtgccga tggacggctg gtacgaatgg 360cgcgtcgacc ccgacgccac cccggggagg ccgaacgcca agacgccgtt cttcctgcac 420cgccacgacg gcgccctgtt gttcacggcc gggctgtggt cggtttggaa gtcttacagg 480tccgccccac cgctgctgag ctgcacggtg atcaccaccg atgccgtggg cgagctggcc 540gagatccatg accggatgcc gctgctgctg gccgaagagg actgggacga ctggctgaat 600ccagacgccc cgccggatcc tgagctgctg gcccgcccgc cggatgtgcg cgacatcgcg 660ctgcgccaag tgtccacgtt ggtcaacaac gtgcgcaaca acgggcctga gctgttggag 720ccggccaggt cgcagcccga gcagatccag ctgctg 75656657DNAMycobacterium tuberculosis 56gtgccagagc tggagacgcc cgacgaccca gagtcgatat accttgcccg cctcgaggat 60gtcggagaac acagaccgac gttcacgggc gacatctacc gactcggcga tggtcgcatg 120gtgatgatcc tccagcaccc atgcgcgctg cggcacggcg ttgacctcca tccgcgactg 180ctggtcgctc ccgtaagacc cgactcgctt cgttccaact gggctagagc cccgttcggc 240acgatgccgc ttccgaagct catcgacggt caggatcact cggcggactt catcaatctt 300gaactcatcg attcaccaac gcttccgacc tgtgagcgga tcgcggtgct cagccagtca 360ggcgtcaact tggtcatgca acggtgggtg taccacagca cccggctcgc cgtgcccacg 420cacacctact ccgacagcac cgttggcccg ttcgatgagg cagacctgat cgaggagtgg 480gtgacggatc gcgtcgacga tggggccgac ccgcaggcgg ccgaacacga atgcgcctcc 540tggctcgatg aaagaatcag cggccgcact cggcgagcgc tgctcagcga ccgtcagcac 600gccagttcaa tacggcgaga agcgcgttct catcgaaagt cggtcaagct ggcggac 65757546DNAMycobacterium tuberculosis 57atggcagact gtgattccgt gactaacagc ccccttgcga ccgctaccgc cacgctgcac 60actaaccgcg gcgacatcaa gatcgccctg ttcggaaacc atgcgcccaa gaccgtcgcc 120aattttgtgg gccttgcgca gggcaccaag gactattcga cccaaaacgc atcaggtggc 180ccgtccggcc cgttctacga cggcgcggtc tttcaccggg tgatccaggg cttcatgatc 240cagggtggcg atccaaccgg gacgggtcgc ggcggacccg gctacaagtt cgccgacgag 300ttccaccccg agctgcaatt cgacaagccc tatctgctcg cgatggccaa cgccggtccg 360ggcaccaacg gctcacagtt tttcatcacc gtcggcaaga ctccgcacct gaaccggcgc 420cacaccattt tcggtgaagt gatcgacgcg gagtcacagc gggttgtgga ggcgatctcc 480aagacggcca ccgacggcaa cgatcggccg acggacccgg tggtgatcga gtcgatcacc 540atctcc 54658849DNAMycobacterium tuberculosis 58atgggggcgc agccgttcat cggcagcgag gcgttggcgg cgggactcat cagctggcat 60gagctgggca agtactacac cgcgatcatg cccaacgtct atctggacaa gcggctgaag 120ccctccctgc ggcaacgcgt tatcgcggcc tggctgtggt cgggccgcaa aggggtgatc 180gccggcgctt cggcatcagc gctgcacggc gcgaaatggg tcgatgacca cgcattggtg 240gagttgatct ggcgcaacgc cagggcgccg aacggggtgc ggactaagga tgagctactg 300ctcgacggcg aagtccagcg cttgtgcggg cttactgtga ctaccgttga acgtacggcc 360ttcgacttgg gcaggcgtcc acccttaggt caggcgataa ccagactgga tgcgcttgcc 420aatgccaccg atttcaagat caacgatgtt agggagctcg cgaggaagca cccccatact 480cgcgggctgc gtcaactaga caaggcgctg gatctcgtcg acccaggtgc gcagtcgccg 540aaggagacgt ggctgcggct cttgctgata aacgccggct ttccacggcc gtccactcag 600atccccttgc tcggcgtcta cgggcatcca
aagtatttcc tcgacatggg atgggaggac 660atcatgctcg cggtcgagta cgacggcgag caacaccgtc tcagccgaga ccagttcgtc 720aaagacgtcg aacgcctgga atacatccgg cgcgccggct ggactcacat cagggtgctg 780gcagaccaca agggacccga cgtcgtccgc cgggttcggc aggcttggga cacgttgaca 840tcacgacgt 84959708DNAMycobacterium tuberculosis 59atgatgcacc gaaccgcact accctcaccg cccgtggcca agcgggtgca gacccgccgg 60gagcaccacg gcgacgtctt tgtcgaccca tatgaatggt tgcgcgacaa ggacagccct 120gaagtaatcg cctacctcga agctgaaaac gactacaccg aacggaccac cgcgcacctt 180gagccattgc ggcaaaagat cttccacgaa atcaaagcgc gtaccaagga aaccgactta 240tcggtgccga cgcgacgtgg caactggtgg tactacgcgc ggacctttga gggaaagcag 300tatggcgtac actgtcgttg cccggtaacc gatcccgacg actggaaccc accagagttc 360gacgagcgca ccgaaatacc cggtgaacag cttctgctcg acgagaacgt ggaagctgac 420ggccacgact tcttcgcact gggcgcggcc agcgtcagcc tggacgataa cctcttagcg 480tattccgttg atgtcgtagg tgacgaacga tataccttgc ggttcaagga tttacgcacc 540ggagaacagt acccggacga gatcgccggg atcggagcgg gagtcacctg ggcagctgac 600aaccactgtc tactacacca ccgtggacgc ggcctggcgt ccggacacag tgtggcgata 660ccgactaggg tccggcgaat cgtcggagcg ggtttaccac gaagccga 70860729DNAMycobacterium tuberculosis 60atgcccaatt tctgggcgtt gccgcccgag atcaactcca cccggatata tctcggcccg 60ggttctggcc cgatactggc cgccgcccag ggatggaacg ctctggccag tgagctggaa 120aagacgaagg tggggttgca gtcagcgctc gacacgttgc tggagtcgta taggggtcag 180tcgtcgcagg ctttgataca gcagaccttg ccgtatgtgc agtggctgac cacgaccgcc 240gagcacgccc ataagaccgc gatccagctc acggcagcgg cgaacgccta cgagcaggct 300agagcggcga tggtgccgcc ggcgatggtg cgcgcgaacc gcgtgcagac cacagtgttg 360aaggcaatca actggttcgg gcaattctcc accaggatcg ccgacaagga ggccgactac 420gaacagatgt ggttccaaga cgcgctagtg atggagaact attgggaagc cgtgcaagag 480gcgatacagt cgacgtcgca ttttgaggat ccaccggaga tggccgacga ctacgacgag 540gcctggatgc tcaacaccgt gttcgactat cacaacgaga acgcaaaaga ggaggtcatc 600catctcgtgc ccgacgtgaa caaggagagg gggcccatcg aactcgtaac caaggtagac 660aaagagggga ccatcagact cgtctacgat ggggagccca cgttttcata caaggaacat 720cctaagttt 72961561DNAMycobacterium tuberculosis 61atggccgacg ctgacaccac cgacttcgac gtcgacgcag aagcaccggg tggaggcgtc 60cgggaggaca cggcgacgga tgctgacgag gccgacgatc aagaagagag attggtcgcc 120gagggcgaga ttgcaggcga ctacctggaa gagttattgg acgtgttgga cttcgatggc 180gacatcgacc tcgatgtcga aggcaatcgt gcggtggtga gcatcgacgg cagtgacgac 240ctgaacaagt tggtcgggcg cgggggcgag gtgctcgacg ctctgcagga actcacccgg 300ttggcggtgc atcagaagac cggtgtgcgg agccggttga tgctagacat cgcgaggtgg 360cgacggcggc gccgggagga attggcggcg ctggccgacg aggtggcgcg gcgagtggcc 420gaaaccggtg accgcgagga actcgttcca atgacgccgt tcgaacggaa gatcgtccac 480gatgcggttg cagcggtgcc aggtgtgcac agcgaaagcg aaggcgtgga gccagaacgc 540cgagtcgttg tgctccgcga c 56162621DNAMycobacterium tuberculosis 62ttgtgtgcaa aaccgtatct aattgatacg attgcgcaca tggctatctg ggatcgcctc 60gtcgaggttg ccgccgagca acatggctac gtcacgactc gcgatgcgcg agacatcggc 120gtcgaccctg tgcagctccg cctcctagcg gggcgcggac gtcttgagcg tgtcggccga 180ggtgtgtacc gggtgcccgt gctgccgcgt ggtgagcacg acgatctcgc agccgcagtg 240tcgtggactt tggggcgtgg cgttatctcg catgagtcgg ccttggcgct tcatgccctc 300gctgacgtga acccgtcgcg catccatctc accgtcccgc gcaacaacca tccgcgtgcg 360gccgggggcg agctgtaccg agttcaccgc cgcgacctcc aggcagccca cgtcacttcg 420gtcgacggaa tacccgtcac gacggttgcg cgcaccatca aagactgcgt gaagacgggc 480acggatcctt atcagcttcg ggccgcgatc gagcgagccg aagccgaggg cacgcttcgt 540cgtgggtcag cagctgagct acgcgctgcg ctcgatgaga ccactgccgg attacgcgct 600cggccgaagc gagcatcggc g 62163555DNAMycobacterium tuberculosis 63atgattgatg aggctctctt cgacgccgaa gagaaaatgg agaaggctgt ggcggtggca 60cgtgacgacc tgtcaactat ccgtaccggc cgcgccaacc ctggcatgtt ctctcggatc 120accatcgact actacggtgc ggccaccccg atcacgcaac tggccagcat caatgtcccc 180gaggcgcggc tagtcgtgat aaagccgtat gaagccaatc agttgcgcgc tatcgagact 240gcaattcgca actccgacct tggagtgaat cccaccaacg acggcgccct tattcgcgtg 300gccgtaccgc agctcaccga agaacgtcgg cgagagctgg tcaaacaggc aaagcataag 360ggggaggagg ccaaggtttc ggtgcgtaat atccgtcgca aagcgatgga ggaactccat 420cgcatccgta aggaaggcga ggccggcgag gatgaggtcg gtcgcgcaga aaaggatctc 480gacaagacca cgcaccaata cgtcacccaa attgatgagc tggttaaaca caaagaaggc 540gagctgctgg aggtc 55564498DNAMycobacterium tuberculosis 64atgcccaagc tcagcgcggg tgtgctgctg tatcgggcgc gcgccggtgt cgtcgacgtc 60cttctggcgc atccgggcgg cccgttttgg gcgggaaagg acgacggcgc ttggtcgatc 120ccgaagggcg aatacaccgg cggcgaagat ccgtggctgg ccgcccggcg cgagttctcc 180gaggagatcg ggttgtgcgt gcctgacggg ccgcgaatcg acttcgggtc gctgaaacag 240tccggcggca aggtggtgac cgtgttcggt gtccgggcgg atctggacat caccgacgca 300cgaagcagca ccttcgaatt ggactggccg aagggctcgg gcaagatgcg taagttcccc 360gaggtcgacc gggtgagctg gtttccggta gcgcgggcac gcaccaaact gctcaagggg 420cagcggggtt ttctcgaccg gttgatggcg cacccggccg tggcgggttt gtctgaagga 480ccagaatccc tgcctcgc 49865144PRTMycobacterium tuberculosis 65Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro1 5 10 15Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg 20 25 30Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu 35 40 45Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys 50 55 60Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu65 70 75 80Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly 85 90 95Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp 100 105 110Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val 115 120 125Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 130 135 14066159PRTMycobacterium tuberculosis 66Val Lys Arg Gly Leu Thr Val Ala Val Ala Gly Ala Ala Ile Leu Val1 5 10 15Ala Gly Leu Ser Gly Cys Ser Ser Asn Lys Ser Thr Thr Gly Ser Gly 20 25 30Glu Thr Thr Thr Ala Ala Gly Thr Thr Ala Ser Pro Gly Ala Ala Ser 35 40 45Gly Pro Lys Val Val Ile Asp Gly Lys Asp Gln Asn Val Thr Gly Ser 50 55 60Val Val Cys Thr Thr Ala Ala Gly Asn Val Asn Ile Ala Ile Gly Gly65 70 75 80Ala Ala Thr Gly Ile Ala Ala Val Leu Thr Asp Gly Asn Pro Pro Glu 85 90 95Val Lys Ser Val Gly Leu Gly Asn Val Asn Gly Val Thr Leu Gly Tyr 100 105 110Thr Ser Gly Thr Gly Gln Gly Asn Ala Ser Ala Thr Lys Asp Gly Ser 115 120 125His Tyr Lys Ile Thr Gly Thr Ala Thr Gly Val Asp Met Ala Asn Pro 130 135 140Met Ser Pro Val Asn Lys Ser Phe Glu Ile Glu Val Thr Cys Ser145 150 15567270PRTMycobacterium tuberculosis 67Met Ala Asn Pro Phe Val Lys Ala Trp Lys Tyr Leu Met Ala Leu Phe1 5 10 15Ser Ser Lys Ile Asp Glu His Ala Asp Pro Lys Val Gln Ile Gln Gln 20 25 30Ala Ile Glu Glu Ala Gln Arg Thr His Gln Ala Leu Thr Gln Gln Ala 35 40 45Ala Gln Val Ile Gly Asn Gln Arg Gln Leu Glu Met Arg Leu Asn Arg 50 55 60Gln Leu Ala Asp Ile Glu Lys Leu Gln Val Asn Val Arg Gln Ala Leu65 70 75 80Thr Leu Ala Asp Gln Ala Thr Ala Ala Gly Asp Ala Ala Lys Ala Thr 85 90 95Glu Tyr Asn Asn Ala Ala Glu Ala Phe Ala Ala Gln Leu Val Thr Ala 100 105 110Glu Gln Ser Val Glu Asp Leu Lys Thr Leu His Asp Gln Ala Leu Ser 115 120 125Ala Ala Ala Gln Ala Lys Lys Ala Val Glu Arg Asn Ala Met Val Leu 130 135 140Gln Gln Lys Ile Ala Glu Arg Thr Lys Leu Leu Ser Gln Leu Glu Gln145 150 155 160Ala Lys Met Gln Glu Gln Val Ser Ala Ser Leu Arg Ser Met Ser Glu 165 170 175Leu Ala Ala Pro Gly Asn Thr Pro Ser Leu Asp Glu Val Arg Asp Lys 180 185 190Ile Glu Arg Arg Tyr Ala Asn Ala Ile Gly Ser Ala Glu Leu Ala Glu 195 200 205Ser Ser Val Gln Gly Arg Met Leu Glu Val Glu Gln Ala Gly Ile Gln 210 215 220Met Ala Gly His Ser Arg Leu Glu Gln Ile Arg Ala Ser Met Arg Gly225 230 235 240Glu Ala Leu Pro Ala Gly Gly Thr Thr Ala Thr Pro Arg Pro Ala Thr 245 250 255Glu Thr Ser Gly Gly Ala Ile Ala Glu Gln Pro Tyr Gly Gln 260 265 27068289PRTMycobacterium tuberculosis 68Met Thr Leu Lys Val Lys Gly Glu Gly Leu Gly Ala Gln Val Thr Gly1 5 10 15Val Asp Pro Lys Asn Leu Asp Asp Ile Thr Thr Asp Glu Ile Arg Asp 20 25 30Ile Val Tyr Thr Asn Lys Leu Val Val Leu Lys Asp Val His Pro Ser 35 40 45Pro Arg Glu Phe Ile Lys Leu Gly Arg Ile Ile Gly Gln Ile Val Pro 50 55 60Tyr Tyr Glu Pro Met Tyr His His Glu Asp His Pro Glu Ile Phe Val65 70 75 80Ser Ser Thr Glu Glu Gly Gln Gly Val Pro Lys Thr Gly Ala Phe Trp 85 90 95His Ile Asp Tyr Met Phe Met Pro Glu Pro Phe Ala Phe Ser Met Val 100 105 110Leu Pro Leu Ala Val Pro Gly His Asp Arg Gly Thr Tyr Phe Ile Asp 115 120 125Leu Ala Arg Val Trp Gln Ser Leu Pro Ala Ala Lys Arg Asp Pro Ala 130 135 140Arg Gly Thr Val Ser Thr His Asp Pro Arg Arg His Ile Lys Ile Arg145 150 155 160Pro Ser Asp Val Tyr Arg Pro Ile Gly Glu Val Trp Asp Glu Ile Asn 165 170 175Arg Thr Thr Pro Pro Ile Lys Trp Pro Thr Val Ile Arg His Pro Lys 180 185 190Thr Gly Gln Glu Ile Leu Tyr Ile Cys Ala Thr Gly Thr Thr Lys Ile 195 200 205Glu Asp Lys Asp Gly Asn Pro Val Asp Pro Glu Val Leu Gln Glu Leu 210 215 220Met Ala Ala Thr Gly Gln Leu Asp Pro Glu Tyr Gln Ser Pro Phe Ile225 230 235 240His Thr Gln His Tyr Gln Val Gly Asp Ile Ile Leu Trp Asp Asn Arg 245 250 255Val Leu Met His Arg Ala Lys His Gly Ser Ala Ala Gly Thr Leu Thr 260 265 270Thr Tyr Arg Leu Thr Met Leu Asp Gly Leu Lys Thr Pro Gly Tyr Ala 275 280 285Ala69199PRTMycobacterium tuberculosis 69Met Ala Glu Asn Ser Asn Ile Asp Asp Ile Lys Ala Pro Leu Leu Ala1 5 10 15Ala Leu Gly Ala Ala Asp Leu Ala Leu Ala Thr Val Asn Glu Leu Ile 20 25 30Thr Asn Leu Arg Glu Arg Ala Glu Glu Thr Arg Thr Asp Thr Arg Ser 35 40 45Arg Val Glu Glu Ser Arg Ala Arg Leu Thr Lys Leu Gln Glu Asp Leu 50 55 60Pro Glu Gln Leu Thr Glu Leu Arg Glu Lys Phe Thr Ala Glu Glu Leu65 70 75 80Arg Lys Ala Ala Glu Gly Tyr Leu Glu Ala Ala Thr Ser Arg Tyr Asn 85 90 95Glu Leu Val Glu Arg Gly Glu Ala Ala Leu Glu Arg Leu Arg Ser Gln 100 105 110Gln Ser Phe Glu Glu Val Ser Ala Arg Ala Glu Gly Tyr Val Asp Gln 115 120 125Ala Val Glu Leu Thr Gln Glu Ala Leu Gly Thr Val Ala Ser Gln Thr 130 135 140Arg Ala Val Gly Glu Arg Ala Ala Lys Leu Val Gly Ile Glu Leu Pro145 150 155 160Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala 165 170 175Pro Ala Lys Lys Ala Ala Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala 180 185 190Ala Lys Lys Val Thr Gln Lys 19570277PRTMycobacterium tuberculosis 70Met Ala Arg Cys Asp Val Leu Val Ser Ala Asp Trp Ala Glu Ser Asn1 5 10 15Leu His Ala Pro Lys Val Val Phe Val Glu Val Asp Glu Asp Thr Ser 20 25 30Ala Tyr Asp Arg Asp His Ile Ala Gly Ala Ile Lys Leu Asp Trp Arg 35 40 45Thr Asp Leu Gln Asp Pro Val Lys Arg Asp Phe Val Asp Ala Gln Gln 50 55 60Phe Ser Lys Leu Leu Ser Glu Arg Gly Ile Ala Asn Glu Asp Thr Val65 70 75 80Ile Leu Tyr Gly Gly Asn Asn Asn Trp Phe Ala Ala Tyr Ala Tyr Trp 85 90 95Tyr Phe Lys Leu Tyr Gly His Glu Lys Val Lys Leu Leu Asp Gly Gly 100 105 110Arg Lys Lys Trp Glu Leu Asp Gly Arg Pro Leu Ser Ser Asp Pro Val 115 120 125Ser Arg Pro Val Thr Ser Tyr Thr Ala Ser Pro Pro Asp Asn Thr Ile 130 135 140Arg Ala Phe Arg Asp Glu Val Leu Ala Ala Ile Asn Val Lys Asn Leu145 150 155 160Ile Asp Val Arg Ser Pro Asp Glu Phe Ser Gly Lys Ile Leu Ala Pro 165 170 175Ala His Leu Pro Gln Glu Gln Ser Gln Arg Pro Gly His Ile Pro Gly 180 185 190Ala Ile Asn Val Pro Trp Ser Arg Ala Ala Asn Glu Asp Gly Thr Phe 195 200 205Lys Ser Asp Glu Glu Leu Ala Lys Leu Tyr Ala Asp Ala Gly Leu Asp 210 215 220Asn Ser Lys Glu Thr Ile Ala Tyr Cys Arg Ile Gly Glu Arg Ser Ser225 230 235 240His Thr Trp Phe Val Leu Arg Glu Leu Leu Gly His Gln Asn Val Lys 245 250 255Asn Tyr Asp Gly Ser Trp Thr Glu Tyr Gly Ser Leu Val Gly Ala Pro 260 265 270Ile Glu Leu Gly Ser 27571210PRTMycobacterium tuberculosis 71Met Thr Lys Pro Thr Ser Ala Gly Gln Ala Asp Asp Ala Leu Val Arg1 5 10 15Leu Ala Arg Glu Arg Phe Asp Leu Pro Asp Gln Val Arg Arg Leu Ala 20 25 30Arg Pro Pro Val Pro Ser Leu Glu Pro Pro Tyr Gly Leu Arg Val Ala 35 40 45Gln Leu Thr Asp Ala Glu Met Leu Ala Glu Trp Met Asn Arg Pro His 50 55 60Leu Ala Ala Ala Trp Glu Tyr Asp Trp Pro Ala Ser Arg Trp Arg Gln65 70 75 80His Leu Asn Ala Gln Leu Glu Gly Thr Tyr Ser Leu Pro Leu Ile Gly 85 90 95Ser Trp His Gly Thr Asp Gly Gly Tyr Leu Glu Leu Tyr Trp Ala Ala 100 105 110Lys Asp Leu Ile Ser His Tyr Tyr Asp Ala Asp Pro Tyr Asp Leu Gly 115 120 125Leu His Ala Ala Ile Ala Asp Leu Ser Lys Val Asn Arg Gly Phe Gly 130 135 140Pro Leu Leu Leu Pro Arg Ile Val Ala Ser Val Phe Ala Asn Glu Pro145 150 155 160Arg Cys Arg Arg Ile Met Phe Asp Pro Asp His Arg Asn Thr Ala Thr 165 170 175Arg Arg Leu Cys Glu Trp Ala Gly Cys Lys Phe Leu Gly Glu His Asp 180 185 190Thr Thr Asn Arg Arg Met Ala Leu Tyr Ala Leu Glu Ala Pro Thr Thr 195 200 205Ala Ala 21072277PRTMycobacterium tuberculosis 72Met Ala Arg Cys Asp Val Leu Val Ser Ala Asp Trp Ala Glu Ser Asn1 5 10 15Leu His Ala Pro Lys Val Val Phe Val Glu Val Asp Glu Asp Thr Ser 20 25 30Ala Tyr Asp Arg Asp His Ile Ala Gly Ala Ile Lys Leu Asp Trp Arg 35 40 45Thr Asp Leu Gln Asp Pro Val Lys Arg Asp Phe Val Asp Ala Gln Gln 50 55 60Phe Ser Lys Leu Leu Ser Glu Arg Gly Ile Ala Asn Glu Asp Thr Val65 70 75 80Ile Leu Tyr Gly Gly Asn Asn Asn Trp Phe Ala Ala Tyr Ala Tyr Trp 85 90 95Tyr Phe Lys Leu Tyr Gly His Glu Lys Val Lys Leu Leu Asp Gly Gly 100 105 110Arg Lys Lys Trp Glu Leu Asp Gly Arg Pro Leu Ser Ser Asp Pro Val 115 120 125Ser Arg Pro Val Thr Ser Tyr Thr Ala Ser Pro Pro Asp Asn Thr Ile 130 135 140Arg Ala Phe Arg Asp Glu Val Leu Ala Ala Ile Asn Val Lys Asn Leu145 150 155 160Ile Asp Val Arg Ser Pro Asp Glu Phe Ser Gly Lys Ile Leu Ala Pro 165 170
175Ala His Leu Pro Gln Glu Gln Ser Gln Arg Pro Gly His Ile Pro Gly 180 185 190Ala Ile Asn Val Pro Trp Ser Arg Ala Ala Asn Glu Asp Gly Thr Phe 195 200 205Lys Ser Asp Glu Glu Leu Ala Lys Leu Tyr Ala Asp Ala Gly Leu Asp 210 215 220Asn Ser Lys Glu Thr Ile Ala Tyr Cys Arg Ile Gly Glu Arg Ser Ser225 230 235 240His Thr Trp Phe Val Leu Arg Glu Leu Leu Gly His Gln Asn Val Lys 245 250 255Asn Tyr Asp Gly Ser Trp Thr Glu Tyr Gly Ser Leu Val Gly Ala Pro 260 265 270Ile Glu Leu Gly Ser 27573195PRTMycobacterium tuberculosis 73Val Ser Asp Glu Asp Arg Thr Asp Arg Ala Thr Glu Asp His Thr Ile1 5 10 15Phe Asp Arg Gly Val Gly Gln Arg Asp Gln Leu Gln Arg Leu Trp Thr 20 25 30Pro Tyr Arg Met Asn Tyr Leu Ala Glu Ala Pro Val Lys Arg Asp Pro 35 40 45Asn Ser Ser Ala Ser Pro Ala Gln Pro Phe Thr Glu Ile Pro Gln Leu 50 55 60Ser Asp Glu Glu Gly Leu Val Val Ala Arg Gly Lys Leu Val Tyr Ala65 70 75 80Val Leu Asn Leu Tyr Pro Tyr Asn Pro Gly His Leu Met Val Val Pro 85 90 95Tyr Arg Arg Val Ser Glu Leu Glu Asp Leu Thr Asp Leu Glu Ser Ala 100 105 110Glu Leu Met Ala Phe Thr Gln Lys Ala Ile Arg Val Ile Lys Asn Val 115 120 125Ser Arg Pro His Gly Phe Asn Val Gly Leu Asn Leu Gly Thr Ser Ala 130 135 140Gly Gly Ser Leu Ala Glu His Leu His Val His Val Val Pro Arg Trp145 150 155 160Gly Gly Asp Ala Asn Phe Ile Thr Ile Ile Gly Gly Ser Lys Val Ile 165 170 175Pro Gln Leu Leu Arg Asp Thr Arg Arg Leu Leu Ala Thr Glu Trp Ala 180 185 190Arg Gln Pro 19574252PRTMycobacterium tuberculosis 74Met Cys Gly Arg Phe Ala Val Thr Thr Asp Pro Ala Gln Leu Ala Glu1 5 10 15Lys Ile Thr Ala Ile Asp Glu Ala Thr Gly Cys Gly Gly Gly Lys Thr 20 25 30Ser Tyr Asn Val Ala Pro Thr Asp Thr Ile Ala Thr Val Val Ser Arg 35 40 45His Ser Glu Pro Asp Asp Glu Pro Thr Arg Arg Val Arg Leu Met Arg 50 55 60Trp Gly Leu Ile Pro Ser Trp Ile Lys Ala Gly Pro Gly Gly Ala Pro65 70 75 80Asp Ala Lys Gly Pro Pro Leu Ile Asn Ala Arg Ala Asp Lys Val Ala 85 90 95Thr Ser Pro Ala Phe Arg Ser Ala Val Arg Ser Lys Arg Cys Leu Val 100 105 110Pro Met Asp Gly Trp Tyr Glu Trp Arg Val Asp Pro Asp Ala Thr Pro 115 120 125Gly Arg Pro Asn Ala Lys Thr Pro Phe Phe Leu His Arg His Asp Gly 130 135 140Ala Leu Leu Phe Thr Ala Gly Leu Trp Ser Val Trp Lys Ser Tyr Arg145 150 155 160Ser Ala Pro Pro Leu Leu Ser Cys Thr Val Ile Thr Thr Asp Ala Val 165 170 175Gly Glu Leu Ala Glu Ile His Asp Arg Met Pro Leu Leu Leu Ala Glu 180 185 190Glu Asp Trp Asp Asp Trp Leu Asn Pro Asp Ala Pro Pro Asp Pro Glu 195 200 205Leu Leu Ala Arg Pro Pro Asp Val Arg Asp Ile Ala Leu Arg Gln Val 210 215 220Ser Thr Leu Val Asn Asn Val Arg Asn Asn Gly Pro Glu Leu Leu Glu225 230 235 240Pro Ala Arg Ser Gln Pro Glu Gln Ile Gln Leu Leu 245 25075219PRTMycobacterium tuberculosis 75Val Pro Glu Leu Glu Thr Pro Asp Asp Pro Glu Ser Ile Tyr Leu Ala1 5 10 15Arg Leu Glu Asp Val Gly Glu His Arg Pro Thr Phe Thr Gly Asp Ile 20 25 30Tyr Arg Leu Gly Asp Gly Arg Met Val Met Ile Leu Gln His Pro Cys 35 40 45Ala Leu Arg His Gly Val Asp Leu His Pro Arg Leu Leu Val Ala Pro 50 55 60Val Arg Pro Asp Ser Leu Arg Ser Asn Trp Ala Arg Ala Pro Phe Gly65 70 75 80Thr Met Pro Leu Pro Lys Leu Ile Asp Gly Gln Asp His Ser Ala Asp 85 90 95Phe Ile Asn Leu Glu Leu Ile Asp Ser Pro Thr Leu Pro Thr Cys Glu 100 105 110Arg Ile Ala Val Leu Ser Gln Ser Gly Val Asn Leu Val Met Gln Arg 115 120 125Trp Val Tyr His Ser Thr Arg Leu Ala Val Pro Thr His Thr Tyr Ser 130 135 140Asp Ser Thr Val Gly Pro Phe Asp Glu Ala Asp Leu Ile Glu Glu Trp145 150 155 160Val Thr Asp Arg Val Asp Asp Gly Ala Asp Pro Gln Ala Ala Glu His 165 170 175Glu Cys Ala Ser Trp Leu Asp Glu Arg Ile Ser Gly Arg Thr Arg Arg 180 185 190Ala Leu Leu Ser Asp Arg Gln His Ala Ser Ser Ile Arg Arg Glu Ala 195 200 205Arg Ser His Arg Lys Ser Val Lys Leu Ala Asp 210 21576182PRTMycobacterium tuberculosis 76Met Ala Asp Cys Asp Ser Val Thr Asn Ser Pro Leu Ala Thr Ala Thr1 5 10 15Ala Thr Leu His Thr Asn Arg Gly Asp Ile Lys Ile Ala Leu Phe Gly 20 25 30Asn His Ala Pro Lys Thr Val Ala Asn Phe Val Gly Leu Ala Gln Gly 35 40 45Thr Lys Asp Tyr Ser Thr Gln Asn Ala Ser Gly Gly Pro Ser Gly Pro 50 55 60Phe Tyr Asp Gly Ala Val Phe His Arg Val Ile Gln Gly Phe Met Ile65 70 75 80Gln Gly Gly Asp Pro Thr Gly Thr Gly Arg Gly Gly Pro Gly Tyr Lys 85 90 95Phe Ala Asp Glu Phe His Pro Glu Leu Gln Phe Asp Lys Pro Tyr Leu 100 105 110Leu Ala Met Ala Asn Ala Gly Pro Gly Thr Asn Gly Ser Gln Phe Phe 115 120 125Ile Thr Val Gly Lys Thr Pro His Leu Asn Arg Arg His Thr Ile Phe 130 135 140Gly Glu Val Ile Asp Ala Glu Ser Gln Arg Val Val Glu Ala Ile Ser145 150 155 160Lys Thr Ala Thr Asp Gly Asn Asp Arg Pro Thr Asp Pro Val Val Ile 165 170 175Glu Ser Ile Thr Ile Ser 18077283PRTMycobacterium tuberculosis 77Met Gly Ala Gln Pro Phe Ile Gly Ser Glu Ala Leu Ala Ala Gly Leu1 5 10 15Ile Ser Trp His Glu Leu Gly Lys Tyr Tyr Thr Ala Ile Met Pro Asn 20 25 30Val Tyr Leu Asp Lys Arg Leu Lys Pro Ser Leu Arg Gln Arg Val Ile 35 40 45Ala Ala Trp Leu Trp Ser Gly Arg Lys Gly Val Ile Ala Gly Ala Ser 50 55 60Ala Ser Ala Leu His Gly Ala Lys Trp Val Asp Asp His Ala Leu Val65 70 75 80Glu Leu Ile Trp Arg Asn Ala Arg Ala Pro Asn Gly Val Arg Thr Lys 85 90 95Asp Glu Leu Leu Leu Asp Gly Glu Val Gln Arg Leu Cys Gly Leu Thr 100 105 110Val Thr Thr Val Glu Arg Thr Ala Phe Asp Leu Gly Arg Arg Pro Pro 115 120 125Leu Gly Gln Ala Ile Thr Arg Leu Asp Ala Leu Ala Asn Ala Thr Asp 130 135 140Phe Lys Ile Asn Asp Val Arg Glu Leu Ala Arg Lys His Pro His Thr145 150 155 160Arg Gly Leu Arg Gln Leu Asp Lys Ala Leu Asp Leu Val Asp Pro Gly 165 170 175Ala Gln Ser Pro Lys Glu Thr Trp Leu Arg Leu Leu Leu Ile Asn Ala 180 185 190Gly Phe Pro Arg Pro Ser Thr Gln Ile Pro Leu Leu Gly Val Tyr Gly 195 200 205His Pro Lys Tyr Phe Leu Asp Met Gly Trp Glu Asp Ile Met Leu Ala 210 215 220Val Glu Tyr Asp Gly Glu Gln His Arg Leu Ser Arg Asp Gln Phe Val225 230 235 240Lys Asp Val Glu Arg Leu Glu Tyr Ile Arg Arg Ala Gly Trp Thr His 245 250 255Ile Arg Val Leu Ala Asp His Lys Gly Pro Asp Val Val Arg Arg Val 260 265 270Arg Gln Ala Trp Asp Thr Leu Thr Ser Arg Arg 275 28078236PRTMycobacterium tuberculosis 78Met Met His Arg Thr Ala Leu Pro Ser Pro Pro Val Ala Lys Arg Val1 5 10 15Gln Thr Arg Arg Glu His His Gly Asp Val Phe Val Asp Pro Tyr Glu 20 25 30Trp Leu Arg Asp Lys Asp Ser Pro Glu Val Ile Ala Tyr Leu Glu Ala 35 40 45Glu Asn Asp Tyr Thr Glu Arg Thr Thr Ala His Leu Glu Pro Leu Arg 50 55 60Gln Lys Ile Phe His Glu Ile Lys Ala Arg Thr Lys Glu Thr Asp Leu65 70 75 80Ser Val Pro Thr Arg Arg Gly Asn Trp Trp Tyr Tyr Ala Arg Thr Phe 85 90 95Glu Gly Lys Gln Tyr Gly Val His Cys Arg Cys Pro Val Thr Asp Pro 100 105 110Asp Asp Trp Asn Pro Pro Glu Phe Asp Glu Arg Thr Glu Ile Pro Gly 115 120 125Glu Gln Leu Leu Leu Asp Glu Asn Val Glu Ala Asp Gly His Asp Phe 130 135 140Phe Ala Leu Gly Ala Ala Ser Val Ser Leu Asp Asp Asn Leu Leu Ala145 150 155 160Tyr Ser Val Asp Val Val Gly Asp Glu Arg Tyr Thr Leu Arg Phe Lys 165 170 175Asp Leu Arg Thr Gly Glu Gln Tyr Pro Asp Glu Ile Ala Gly Ile Gly 180 185 190Ala Gly Val Thr Trp Ala Ala Asp Asn His Cys Leu Leu His His Arg 195 200 205Gly Arg Gly Leu Ala Ser Gly His Ser Val Ala Ile Pro Thr Arg Val 210 215 220Arg Arg Ile Val Gly Ala Gly Leu Pro Arg Ser Arg225 230 23579243PRTMycobacterium tuberculosis 79Met Pro Asn Phe Trp Ala Leu Pro Pro Glu Ile Asn Ser Thr Arg Ile1 5 10 15Tyr Leu Gly Pro Gly Ser Gly Pro Ile Leu Ala Ala Ala Gln Gly Trp 20 25 30Asn Ala Leu Ala Ser Glu Leu Glu Lys Thr Lys Val Gly Leu Gln Ser 35 40 45Ala Leu Asp Thr Leu Leu Glu Ser Tyr Arg Gly Gln Ser Ser Gln Ala 50 55 60Leu Ile Gln Gln Thr Leu Pro Tyr Val Gln Trp Leu Thr Thr Thr Ala65 70 75 80Glu His Ala His Lys Thr Ala Ile Gln Leu Thr Ala Ala Ala Asn Ala 85 90 95Tyr Glu Gln Ala Arg Ala Ala Met Val Pro Pro Ala Met Val Arg Ala 100 105 110Asn Arg Val Gln Thr Thr Val Leu Lys Ala Ile Asn Trp Phe Gly Gln 115 120 125Phe Ser Thr Arg Ile Ala Asp Lys Glu Ala Asp Tyr Glu Gln Met Trp 130 135 140Phe Gln Asp Ala Leu Val Met Glu Asn Tyr Trp Glu Ala Val Gln Glu145 150 155 160Ala Ile Gln Ser Thr Ser His Phe Glu Asp Pro Pro Glu Met Ala Asp 165 170 175Asp Tyr Asp Glu Ala Trp Met Leu Asn Thr Val Phe Asp Tyr His Asn 180 185 190Glu Asn Ala Lys Glu Glu Val Ile His Leu Val Pro Asp Val Asn Lys 195 200 205Glu Arg Gly Pro Ile Glu Leu Val Thr Lys Val Asp Lys Glu Gly Thr 210 215 220Ile Arg Leu Val Tyr Asp Gly Glu Pro Thr Phe Ser Tyr Lys Glu His225 230 235 240Pro Lys Phe80187PRTMycobacterium tuberculosis 80Met Ala Asp Ala Asp Thr Thr Asp Phe Asp Val Asp Ala Glu Ala Pro1 5 10 15Gly Gly Gly Val Arg Glu Asp Thr Ala Thr Asp Ala Asp Glu Ala Asp 20 25 30Asp Gln Glu Glu Arg Leu Val Ala Glu Gly Glu Ile Ala Gly Asp Tyr 35 40 45Leu Glu Glu Leu Leu Asp Val Leu Asp Phe Asp Gly Asp Ile Asp Leu 50 55 60Asp Val Glu Gly Asn Arg Ala Val Val Ser Ile Asp Gly Ser Asp Asp65 70 75 80Leu Asn Lys Leu Val Gly Arg Gly Gly Glu Val Leu Asp Ala Leu Gln 85 90 95Glu Leu Thr Arg Leu Ala Val His Gln Lys Thr Gly Val Arg Ser Arg 100 105 110Leu Met Leu Asp Ile Ala Arg Trp Arg Arg Arg Arg Arg Glu Glu Leu 115 120 125Ala Ala Leu Ala Asp Glu Val Ala Arg Arg Val Ala Glu Thr Gly Asp 130 135 140Arg Glu Glu Leu Val Pro Met Thr Pro Phe Glu Arg Lys Ile Val His145 150 155 160Asp Ala Val Ala Ala Val Pro Gly Val His Ser Glu Ser Glu Gly Val 165 170 175Glu Pro Glu Arg Arg Val Val Val Leu Arg Asp 180 18581207PRTMycobacterium tuberculosis 81Leu Cys Ala Lys Pro Tyr Leu Ile Asp Thr Ile Ala His Met Ala Ile1 5 10 15Trp Asp Arg Leu Val Glu Val Ala Ala Glu Gln His Gly Tyr Val Thr 20 25 30Thr Arg Asp Ala Arg Asp Ile Gly Val Asp Pro Val Gln Leu Arg Leu 35 40 45Leu Ala Gly Arg Gly Arg Leu Glu Arg Val Gly Arg Gly Val Tyr Arg 50 55 60Val Pro Val Leu Pro Arg Gly Glu His Asp Asp Leu Ala Ala Ala Val65 70 75 80Ser Trp Thr Leu Gly Arg Gly Val Ile Ser His Glu Ser Ala Leu Ala 85 90 95Leu His Ala Leu Ala Asp Val Asn Pro Ser Arg Ile His Leu Thr Val 100 105 110Pro Arg Asn Asn His Pro Arg Ala Ala Gly Gly Glu Leu Tyr Arg Val 115 120 125His Arg Arg Asp Leu Gln Ala Ala His Val Thr Ser Val Asp Gly Ile 130 135 140Pro Val Thr Thr Val Ala Arg Thr Ile Lys Asp Cys Val Lys Thr Gly145 150 155 160Thr Asp Pro Tyr Gln Leu Arg Ala Ala Ile Glu Arg Ala Glu Ala Glu 165 170 175Gly Thr Leu Arg Arg Gly Ser Ala Ala Glu Leu Arg Ala Ala Leu Asp 180 185 190Glu Thr Thr Ala Gly Leu Arg Ala Arg Pro Lys Arg Ala Ser Ala 195 200 20582185PRTMycobacterium tuberculosis 82Met Ile Asp Glu Ala Leu Phe Asp Ala Glu Glu Lys Met Glu Lys Ala1 5 10 15Val Ala Val Ala Arg Asp Asp Leu Ser Thr Ile Arg Thr Gly Arg Ala 20 25 30Asn Pro Gly Met Phe Ser Arg Ile Thr Ile Asp Tyr Tyr Gly Ala Ala 35 40 45Thr Pro Ile Thr Gln Leu Ala Ser Ile Asn Val Pro Glu Ala Arg Leu 50 55 60Val Val Ile Lys Pro Tyr Glu Ala Asn Gln Leu Arg Ala Ile Glu Thr65 70 75 80Ala Ile Arg Asn Ser Asp Leu Gly Val Asn Pro Thr Asn Asp Gly Ala 85 90 95Leu Ile Arg Val Ala Val Pro Gln Leu Thr Glu Glu Arg Arg Arg Glu 100 105 110Leu Val Lys Gln Ala Lys His Lys Gly Glu Glu Ala Lys Val Ser Val 115 120 125Arg Asn Ile Arg Arg Lys Ala Met Glu Glu Leu His Arg Ile Arg Lys 130 135 140Glu Gly Glu Ala Gly Glu Asp Glu Val Gly Arg Ala Glu Lys Asp Leu145 150 155 160Asp Lys Thr Thr His Gln Tyr Val Thr Gln Ile Asp Glu Leu Val Lys 165 170 175His Lys Glu Gly Glu Leu Leu Glu Val 180 18583166PRTMycobacterium tuberculosis 83Met Pro Lys Leu Ser Ala Gly Val Leu Leu Tyr Arg Ala Arg Ala Gly1 5 10 15Val Val Asp Val Leu Leu Ala His Pro Gly Gly Pro Phe Trp Ala Gly 20 25 30Lys Asp Asp Gly Ala Trp Ser Ile Pro Lys Gly Glu Tyr Thr Gly Gly 35 40 45Glu Asp Pro Trp Leu Ala Ala Arg Arg Glu Phe Ser Glu Glu Ile Gly 50 55 60Leu Cys Val Pro Asp Gly Pro Arg Ile Asp Phe Gly Ser Leu Lys Gln65 70 75 80Ser Gly Gly Lys Val Val Thr Val Phe Gly Val Arg Ala Asp Leu Asp 85 90 95Ile Thr Asp Ala Arg Ser Ser Thr Phe Glu Leu Asp Trp Pro Lys Gly 100 105 110Ser Gly Lys Met Arg Lys Phe Pro Glu Val Asp Arg Val Ser Trp Phe 115 120 125Pro Val Ala Arg Ala Arg Thr Lys Leu Leu Lys Gly Gln Arg Gly Phe
130 135 140Leu Asp Arg Leu Met Ala His Pro Ala Val Ala Gly Leu Ser Glu Gly145 150 155 160Pro Glu Ser Leu Pro Arg 1658435DNAArtificial SequenceSynthetic Oligonucleotide Primer 84gaaggagata taccatgcat catcatcatc atcat 358520DNAArtificial SequenceSynthetic Oligonucleotide Primer 85tgatgatgag aacccccccc 208650DNAArtificial SequenceSynthetic Oligonucleotide Primer 86gaaggagata taccatgcat catcatcatc atcatgtgag cgcttcggag 508740DNAArtificial SequenceSynthetic Oligonucleotide Primer 87tgatgatgag aacccccccc aggacctcca tgccggcgca 408850DNAArtificial SequenceSynthetic Oligonucleotide Primer 88gaaggagata taccatgcat catcatcatc atcatatgct gccgaagaac 508940DNAArtificial SequenceSynthetic Oligonucleotide Primer 89tgatgatgag aacccccccc gccctcggcg gcgtctttcg 409050DNAArtificial SequenceSynthetic Oligonucleotide Primer 90gaaggagata taccatgcat catcatcatc atcatgtgag agttttgttg 509140DNAArtificial SequenceSynthetic Oligonucleotide Primer 91tgatgatgag aacccccccc ctttcccaga gcccgcaacg 409250DNAArtificial SequenceSynthetic Oligonucleotide Primer 92gaaggagata taccatgcat catcatcatc atcatgtggt tatgcctctt 509340DNAArtificial SequenceSynthetic Oligonucleotide Primer 93tgatgatgag aacccccccc tcccgaccct tcgggctggt 409450DNAArtificial SequenceSynthetic Oligonucleotide Primer 94gaaggagata taccatgcat catcatcatc atcatatgtc ggctcccgaa 509540DNAArtificial SequenceSynthetic Oligonucleotide Primer 95tgatgatgag aacccccccc ggcggtcacc agcgagtagc 409650DNAArtificial SequenceSynthetic Oligonucleotide Primer 96gaaggagata taccatgcat catcatcatc atcatatgct cgagaaggcc 509740DNAArtificial SequenceSynthetic Oligonucleotide Primer 97tgatgatgag aacccccccc gtcgaactga ggcggctcgg 409850DNAArtificial SequenceSynthetic Oligonucleotide Primer 98gaaggagata taccatgcat catcatcatc atcatatgcc atccgacacc 509940DNAArtificial SequenceSynthetic Oligonucleotide Primer 99tgatgatgag aacccccccc cgtttccttc cgagttccaa 4010050DNAArtificial SequenceSynthetic Oligonucleotide Primer 100gaaggagata taccatgcat catcatcatc atcatgtgga cgagatcctg 5010140DNAArtificial SequenceSynthetic Oligonucleotide Primer 101tgatgatgag aacccccccc cctcgctcgg cgggccagtc 4010250DNAArtificial SequenceSynthetic Oligonucleotide Primer 102gaaggagata taccatgcat catcatcatc atcatatgac tacgagctac 5010340DNAArtificial SequenceSynthetic Oligonucleotide Primer 103tgatgatgag aacccccccc aacagccgcg agttcatggt 4010450DNAArtificial SequenceSynthetic Oligonucleotide Primer 104gaaggagata taccatgcat catcatcatc atcatatgag cgtggattac 5010540DNAArtificial SequenceSynthetic Oligonucleotide Primer 105tgatgatgag aacccccccc gctgaactga gtgtgcggcc 4010650DNAArtificial SequenceSynthetic Oligonucleotide Primer 106gaaggagata taccatgcat catcatcatc atcatatgca gacaacccca 5010740DNAArtificial SequenceSynthetic Oligonucleotide Primer 107tgatgatgag aacccccccc acgcgccacc gctttggccc 4010850DNAArtificial SequenceSynthetic Oligonucleotide Primer 108gaaggagata taccatgcat catcatcatc atcatatgac gatccctgat 5010940DNAArtificial SequenceSynthetic Oligonucleotide Primer 109tgatgatgag aacccccccc caggccatca aaaaagtcct 40110501DNAMycobacterium tuberculosis 110gtgagcgctt cggagttctc ccgtgctgaa ctcgccgccg ccttcgagaa gttcgagaag 60accgtggccc gcgccgccgc gacgcgcgac tgggattgct gggtgcagca ctacaccccc 120gacgtcgaat acatcgagca cgcggcgggc atcatgcgag gccgccagcg ggtacgtgcc 180tggattcaag aaacgatgac gaccttcccg ggcagtcaca tggtggcctt cccgtcgctg 240tggtcggtga tcgacgagtc caccgggcga attatctgcg aattggacaa ccccatgctc 300gaccccggcg acggcagcgt gatcagcgcg acgaacattt cgatcatcac ctatgccggc 360aatggccagt ggtgccgtca agaagacatc tacaacccgt tgcggttcct gcgggcggcg 420atgaagtggt gtcgcaaggc gcaggagttg ggcaccctcg acgaggacgc ggcgcgttgg 480atgcgccggc atggaggtcc t 501111525DNAMycobacterium tuberculosis 111atgctgccga agaacaccag acccacctcg gaaaccgccg aagagttctg ggacaactcg 60ctgtggtgca gctggggcga ccgagaaacg ggatacaccc gcaccgtcac ggtttcgatc 120tgccaggtgg cggacggcga acgtgaggcc gaaggggttc gggacatgat gcggctggag 180tgtccggctg ggctggatct acggacaccc aacccggagg catacgagat taccggtcag 240cggcccggag aattcgtgtt cgtgctcggc tatctggggc atgtgcgggc catcgtgggc 300aactgttaca tcgagatcat gccgatgggc accagggtcg agctgagcaa gttggccgat 360gtggcattgg atatcggccg cagtgtcgga tgctcggcct acgagaacga cttcacgctg 420ccggacattc caacgcagtg gcgcaaccag ccgctgggct ggtacacgca aggccttgcc 480ccctacctgc cggggctgtc ggacccgaaa gacgccgccg agggc 525112543DNAMycobacterium tuberculosis 112gtgagagttt 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 540aag 543113564DNAMycobacterium tuberculosis 113gtggttatgc ctcttgtcac gccaaccacc gcggttccat caccgggacc cacacggctg 60cgtgtagccg atctcctgcg cgccaccgac caagccgcag acgacgtgct tggcgggcgc 120tgcgaccacc tgctacccga cggtggtgtc ccgcagacgc agcgctggta cacccgcatc 180cacggtgacg aggagctgga tatctggctg attagctggg ttcccggtca accgaccgag 240ctgcacgacc atggcgggtc cctgggagcg ttgaccgtgc tgagcgggtc gctcaacgaa 300tatcgttggg acggccgtcg gttgcgacgg cgccgcctcg atgccggtga tcaggcaggg 360ttcccgttgg gttgggtgca cgacgtggtg tgggcgcccc ggccgattgg ggggcctgat 420gcggccggga tggctgtggc gccaaccctg agcgtgcacg cctactcgcc gccgctgacg 480gcgatgtcgt actacgagat caccgaacgc aacacgctgc gccgccagcg caccgaattg 540accgaccagc ccgaagggtc ggga 564114621DNAMycobacterium tuberculosis 114atgtcggctc ccgaacgggt aaccggcttg tccgggcaac gttacgggga agtccttctc 60gtaacacccg gggaggccgg tccacaggcc accgtttaca acagcttccc gcttaacgat 120tgtccggccg agctgtggtc cgcgctcgat ccgcaagccc tagccaccga acacaaagcg 180gccaccgccc tgctcaacgg tccgcgctat tggttgatga acgccatcga gaaggcgccc 240cagggcccgc cggtgacgaa gaccttcggc gggatcgaga tgctccagca ggccacggtg 300ctgctgtcat cgatgaaccc tgccccatac accgtcagcc aggtcagccg caacacggtc 360tttgtgttca acgccggcga agaggtctac gaactgcagg accccaaggg acagcgctgg 420gtgatgcaga cgtggagtca agtggtggac cccaacctgt cccgagccga cctgcccaag 480ctgggtgaac ggctcaacct gccagccggg tggtcctatc atacccgcgt gcttaccagc 540gagttgcggg tcgacactac caaccgggag gcccgcgtcc tgcaagacga cctcaccaac 600agctactcgc tggtgaccgc c 621115621DNAMycobacterium tuberculosis 115atgctcgaga aggcccccca gaagtctgtc gccgatttct ggttcgatcc gctgtgcccg 60tggtgctgga tcacgtcgcg ctggatcctc gaggtggcaa aggtccgcga catcgaggtg 120aacttccacg tcatgagcct ggcaatactc aacgaaaacc gtgacgacct gcccgagcaa 180taccgcgaag gcatggcgag ggcatgggga ccggtacggg tggcgatcgc cgccgagcag 240gcccatgggg cgaaagtcct ggacccgctg tacaccgcga tgggcaaccg gattcacaac 300cagggcaacc acgaactcga cgaggtcatc acccagtcgc tggcggacgc cggtctgccc 360gcggagttgg ccaaggccgc taccagcgac gcttacgaca acgccctgcg caaaagccac 420cacgccggga tggacgcggt gggcgaggac gtcggtacgc cgacgatcca tgtcaatggt 480gtggcgttct tcgggccggt gctctcgaag attccgcgcg gcgaggaagc cggcaagctc 540tgggatgcct cggttacctt cgcttcctac ccgcactttt ttgagctcaa gcggacccgc 600accgagccgc ctcagttcga c 621116642DNAMycobacterium tuberculosis 116atgccatccg acaccagccc caacgggcta agccgccgtg aggagttgct ggctgttgcc 60accaaactat tcgcggcgcg cggttatcac ggcacccgga tggacgacgt cgccgatgtg 120atcgggctca acaaagcaac ggtctatcac tactacgcca gcaagtcgct gatcctgttc 180gacatttacc gtcaggcggc cgagggcacc ctggccgccg tgcacgacga tccgtcctgg 240acggcccgtg aagcgctgta ccagtacacg gtccggctgc tcactgcgat cgcgagcaac 300cccgagcggg ccgccgtgta cttccaggag cagccctaca tcaccgagtg gttcaccagc 360gagcaggtcg ccgaggtccg cgagaaggag cagcaagtct acgagcacgt acacggcctg 420atcgaccgcg ggattgccag cggcgagttc tatgagtgcg actcgcatgt ggtggcgctg 480gggtacatcg ggatgacgct gggcagctac cgctggctgc ggccgagcgg gcgccgaacg 540gccaaggaga tcgcggcgga gttcagcacg gcactgctgc gcgggctgat ccgcgacgaa 600tcgatccgca accagtctcc gcttggaact cggaaggaaa cg 642117672DNAMycobacterium tuberculosis 117gtggacgaga tcctggccag ggcaggaatc ttccaaggcg tggagcccag cgcaatcgcc 60gcactgacga aacagctgca gcccgtcgac ttcccccgtg gacacacggt cttcgcggaa 120ggggagccgg gcgatcggct gtacatcatc atctcgggga aggtcaagat cggtcgccgg 180gcaccagacg gccgagaaaa cctgttaacc atcatgggcc cgtcggacat gttcggcgag 240ttgtcgatct tcgacccggg tccgcgcacg tccagcgcga ccacgatcac cgaggtgcgg 300gcggtgtcga tggaccgcga cgcgctgcgg tcatggatcg ccgatcgtcc cgaaatctcc 360gaacagctgc tgcgggtgct ggcccgccgg ctgcgccgca ccaacaacaa cctggccgac 420ctcatcttca ccgatgtgcc cggtcgggtg gccaagcagc tgttgcagct cgcccagcgt 480ttcggcaccc aggaaggtgg cgcattgcgg gtcacccacg acctgacaca ggaagaaatc 540gcccagctgg tcggggcctc acgcgagacg gtgaacaagg cactggctga tttcgctcac 600cgcggctgga tccgccttga gggcaagagt gtgctgatct ctgactccga aagactggcc 660cgccgagcga gg 672118708DNAMycobacterium tuberculosis 118atgactacga gctacgccaa gatcgagata accgggacac tgaccgtcct gacgggcctg 60cagatcgggg ccggcgatgg cttctccgcc atcggcgcgg tcgacaagcc tgtcgttcgt 120gatccgctga gcaggctgcc gatgattccg ggtaccagcc tgaagggcaa ggtccgcacc 180ttgctgtccc gccaatacgg cgccgacaca gaaacgtttt acaggaagcc gaatgaggac 240cacgcccata tccgtcggct tttcggcgac accgaggagt acatgacggg ccgactcgtc 300ttccgcgaca cgaagctcac caacaaagac gacctcgaag cccgcggcgc taagactctc 360accgaggtga aattcgagaa cgccatcaac cgggtgaccg caaaggcaaa ccttcgccag 420atggaacgcg tgatccccgg cagcgagttc gcgttctcac ttgtctacga ggtctccttc 480ggcacccccg gcgaggaaca gaaggcgtct ctgccttcct ccgatgagat catcgaggac 540ttcaacgcca tcgcgcgcgg cctgaagttg ctcgaactcg actacctcgg cggcagcgga 600acccgtggct acgggcaggt caagttcagc aacctgaaag cccgcgccgc agtcggcgcc 660ctcgacggtt ctctgctgga gaagctaaac catgaactcg cggctgtt 708119762DNAMycobacterium tuberculosis 119atgagcgtgg attaccccca aatggctgct acccggggaa gaatagaacc ggccccgcgg 60cgagttcgcg gctatctcgg acatgtgctc gtcttcgaca ccagtgcggc gcgctatgtc 120tgggaggttc cctactaccc gcagtactac atcccgctgg cggatgtccg catggagttc 180ctgcgcgacg agaaccaccc gcagcgagtg cagctgggtc cgtcgcggct gcactccttg 240gtaagcgccg gtcagaccca ccgatcggcg gcgcgggtat tcgatgtcga cggcgacagc 300ccggtggcgg gcaccgtgcg tttcaactgg gatccgctgc ggtggttcga ggaggacgag 360ccgatctacg gccatccgcg caatccctat cagcgggccg atgcgctgcg ctcgcaccga 420cacgtccgtg tcgagctgga cggcattgtg ctcgctgaca cccgatcgcc cgttctgcta 480ttcgaaactg ggatacccac aaggtattac atcgatccgg ccgacatcgc tttcgagcat 540ctggagccca cctcgacgca gacgttgtgt ccgtacaagg ggacgacgtc gggctattgg 600tctgtgcgcg tcggcgacgc cgtgcaccgc gacctggcct ggacgtatca ctatccactg 660cccgccgttg ccccgatcgc cggcctggtg gcgttttaca acgagaaggt cgacctcacc 720gtcgacggcg tcgccctgcc gcggccgcac actcagttca gc 762120783DNAMycobacterium tuberculosis 120atgcagacaa ccccaggcaa gcgtcaacga cggcagcgcg gatccatcaa ccccgaggac 60atcatcagcg gcgcattcga actcgcccag caggtatcga tagacaactt gagcatgcca 120ttgctcggca aacaccttgg cgtcggggtc accagcatct actggtactt ccgcaagaag 180gacgatctgc tcaacgcgat gaccgaccgc gctttgagca agtacgtgtt cgctaccccg 240tacatcgaag ccggcgactg gcgcgaaacg ttgcgcaatc atgcccgctc gatgcggaag 300acgttcgcgg acaaccccgt actgtgcgat ctgatactga ttcgagcggc gctgtccccg 360aaaacggcgc ggttgggcgc ccaagagatg gagaaggcca tcgccaatct ggtgacggcg 420ggcctgtcgc tcgaagacgc tttcgacatc tactcggcgg tttcggtcca cgtgcgcgga 480tcggtggtgc tagatcggct ctcccgcaag agccagtcgg cgggcagcgg accatccgcc 540attgaacacc ccgtggccat cgatcccgcg acgactccgc tgcttgctca cgcaactggg 600agggggcatc ggatcggggc ccccgatgaa accaatttcg aatatggtct cgaatgcatc 660ctcgaccatg ctggccggtt gatcgaacaa agctcgaaag ccgctggtga ggtcgcagtg 720cgccgcccca cggccaccgc cgatgcgcct acgccgggcg cgcgggccaa agcggtggcg 780cgt 783121918DNAMycobacterium tuberculosis 121atgacgatcc ctgatgccca gacgttgatg cggccgattc tcgcgtatct tgccgatgga 60caagcgaagt cggccaagga cgtcatcgcg gcgatgtccg acgagttcgg tctgtccgac 120gacgagcggg cgcagatgtt gcccagcggt cggcaaagga ccatgtacga cagggtgcac 180tggtctctca ctcacatgtc gcaggccgga ttgctcgacc gtcccacgcg gggccacgtc 240caggtcacgg acacgggccg tcaagtcctg aaggcgcatc ccgagcgcgt cgacatggct 300gtgctgcggg agttcccgtc gtacatcgct tttcgtgagc gaaccaaagc caagcagcca 360gtcgacgcga ccgccaagcg accgtccggg gacgatgtgc aggtctcacc cgaggatctc 420atcgacgctg cgcttgcgga gaaccgggca gccgtcgagg gggagatcct gaagaaggca 480ctcacgttgt cgcccaccgg gtttgaagat ctggttatca gacttttgga ggcgatgggt 540tacgggcgag ccggcgcggt ggaacggacg agtgcctccg gtgacgctgg catcgacgga 600atcatcagcc aggacccgct cgggctggac cgcatctacg tgcaggccaa gcgatacgcc 660gtcgaccaaa cgattggccg gccgaagatc cacgagttcg ccggcgccct cctgggcaag 720cagggcgacc ggggcgtcta catcaccacg tcatcgtttt cccgcggtgc ccgcgaggaa 780gctgagcgga tcaacgcccg gatcgaactc atcgacggcg ctcggctggc cgagctgctc 840gtgcggtatc gagtcggtgt ccaggcggtg cagaccgtcg aactcttacg gctcgacgag 900gacttttttg atggcctg 918122167PRTMycobacterium tuberculosis 122Val Ser Ala Ser Glu Phe Ser Arg Ala Glu Leu Ala Ala Ala Phe Glu1 5 10 15Lys Phe Glu Lys Thr Val Ala Arg Ala Ala Ala Thr Arg Asp Trp Asp 20 25 30Cys Trp Val Gln His Tyr Thr Pro Asp Val Glu Tyr Ile Glu His Ala 35 40 45Ala Gly Ile Met Arg Gly Arg Gln Arg Val Arg Ala Trp Ile Gln Glu 50 55 60Thr Met Thr Thr Phe Pro Gly Ser His Met Val Ala Phe Pro Ser Leu65 70 75 80Trp Ser Val Ile Asp Glu Ser Thr Gly Arg Ile Ile Cys Glu Leu Asp 85 90 95Asn Pro Met Leu Asp Pro Gly Asp Gly Ser Val Ile Ser Ala Thr Asn 100 105 110Ile Ser Ile Ile Thr Tyr Ala Gly Asn Gly Gln Trp Cys Arg Gln Glu 115 120 125Asp Ile Tyr Asn Pro Leu Arg Phe Leu Arg Ala Ala Met Lys Trp Cys 130 135 140Arg Lys Ala Gln Glu Leu Gly Thr Leu Asp Glu Asp Ala Ala Arg Trp145 150 155 160Met Arg Arg His Gly Gly Pro 165123175PRTMycobacterium tuberculosis 123Met Leu Pro Lys Asn Thr Arg Pro Thr Ser Glu Thr Ala Glu Glu Phe1 5 10 15Trp Asp Asn Ser Leu Trp Cys Ser Trp Gly Asp Arg Glu Thr Gly Tyr 20 25 30Thr Arg Thr Val Thr Val Ser Ile Cys Gln Val Ala Asp Gly Glu Arg 35 40 45Glu Ala Glu Gly Val Arg Asp Met Met Arg Leu Glu Cys Pro Ala Gly 50 55 60Leu Asp Leu Arg Thr Pro Asn Pro Glu Ala Tyr Glu Ile Thr Gly Gln65 70 75 80Arg Pro Gly Glu Phe Val Phe Val Leu Gly Tyr Leu Gly His Val Arg 85 90 95Ala Ile Val Gly Asn Cys Tyr Ile Glu Ile Met Pro Met Gly Thr Arg 100 105 110Val Glu Leu Ser Lys Leu Ala Asp Val Ala Leu Asp Ile Gly Arg Ser 115 120 125Val Gly Cys Ser Ala Tyr Glu Asn Asp Phe Thr Leu Pro Asp Ile Pro 130 135 140Thr Gln Trp Arg Asn Gln Pro Leu Gly Trp Tyr Thr Gln Gly Leu Ala145 150 155 160Pro Tyr Leu Pro Gly Leu Ser Asp Pro Lys Asp Ala Ala Glu Gly 165 170 175124181PRTMycobacterium tuberculosis 124Val 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 180125188PRTMycobacterium tuberculosis 125Val Val Met Pro Leu Val Thr Pro Thr Thr Ala Val Pro Ser Pro Gly1 5 10 15Pro Thr Arg Leu Arg Val Ala Asp Leu Leu Arg Ala Thr Asp Gln Ala 20 25 30Ala Asp Asp Val Leu Gly Gly Arg Cys Asp His Leu Leu Pro Asp Gly 35 40 45Gly Val Pro Gln Thr Gln Arg Trp Tyr Thr Arg Ile His Gly Asp Glu 50 55 60Glu Leu Asp Ile Trp Leu Ile Ser Trp Val Pro Gly Gln Pro Thr Glu65 70 75 80Leu His Asp His Gly Gly Ser Leu Gly Ala Leu Thr Val Leu Ser Gly 85 90 95Ser Leu Asn Glu Tyr Arg Trp Asp Gly Arg Arg Leu Arg Arg Arg Arg 100 105 110Leu Asp Ala Gly Asp Gln Ala Gly Phe Pro Leu Gly Trp Val His Asp 115 120 125Val Val Trp Ala Pro Arg Pro Ile Gly Gly Pro Asp Ala Ala Gly Met 130 135 140Ala Val Ala Pro Thr Leu Ser Val His Ala Tyr Ser Pro Pro Leu Thr145 150 155 160Ala Met Ser Tyr Tyr Glu Ile Thr Glu Arg Asn Thr Leu Arg Arg Gln 165 170 175Arg Thr Glu Leu Thr Asp Gln Pro Glu Gly Ser Gly 180 185126207PRTMycobacterium tuberculosis 126Met Ser Ala Pro Glu Arg Val Thr Gly Leu Ser Gly Gln Arg Tyr Gly1 5 10 15Glu Val Leu Leu Val Thr Pro Gly Glu Ala Gly Pro Gln Ala Thr Val 20 25 30Tyr Asn Ser Phe Pro Leu Asn Asp Cys Pro Ala Glu Leu Trp Ser Ala 35 40 45Leu Asp Pro Gln Ala Leu Ala Thr Glu His Lys Ala Ala Thr Ala Leu 50 55 60Leu Asn Gly Pro Arg Tyr Trp Leu Met Asn Ala Ile Glu Lys Ala Pro65 70 75 80Gln Gly Pro Pro Val Thr Lys Thr Phe Gly Gly Ile Glu Met Leu Gln 85 90 95Gln Ala Thr Val Leu Leu Ser Ser Met Asn Pro Ala Pro Tyr Thr Val 100 105 110Ser Gln Val Ser Arg Asn Thr Val Phe Val Phe Asn Ala Gly Glu Glu 115 120 125Val Tyr Glu Leu Gln Asp Pro Lys Gly Gln Arg Trp Val Met Gln Thr 130 135 140Trp Ser Gln Val Val Asp Pro Asn Leu Ser Arg Ala Asp Leu Pro Lys145 150 155 160Leu Gly Glu Arg Leu Asn Leu Pro Ala Gly Trp Ser Tyr His Thr Arg 165 170 175Val Leu Thr Ser Glu Leu Arg Val Asp Thr Thr Asn Arg Glu Ala Arg 180 185 190Val Leu Gln Asp Asp Leu Thr Asn Ser Tyr Ser Leu Val Thr Ala 195 200 205127207PRTMycobacterium tuberculosis 127Met Leu Glu Lys Ala Pro Gln Lys Ser Val Ala Asp Phe Trp Phe Asp1 5 10 15Pro Leu Cys Pro Trp Cys Trp Ile Thr Ser Arg Trp Ile Leu Glu Val 20 25 30Ala Lys Val Arg Asp Ile Glu Val Asn Phe His Val Met Ser Leu Ala 35 40 45Ile Leu Asn Glu Asn Arg Asp Asp Leu Pro Glu Gln Tyr Arg Glu Gly 50 55 60Met Ala Arg Ala Trp Gly Pro Val Arg Val Ala Ile Ala Ala Glu Gln65 70 75 80Ala His Gly Ala Lys Val Leu Asp Pro Leu Tyr Thr Ala Met Gly Asn 85 90 95Arg Ile His Asn Gln Gly Asn His Glu Leu Asp Glu Val Ile Thr Gln 100 105 110Ser Leu Ala Asp Ala Gly Leu Pro Ala Glu Leu Ala Lys Ala Ala Thr 115 120 125Ser Asp Ala Tyr Asp Asn Ala Leu Arg Lys Ser His His Ala Gly Met 130 135 140Asp Ala Val Gly Glu Asp Val Gly Thr Pro Thr Ile His Val Asn Gly145 150 155 160Val Ala Phe Phe Gly Pro Val Leu Ser Lys Ile Pro Arg Gly Glu Glu 165 170 175Ala Gly Lys Leu Trp Asp Ala Ser Val Thr Phe Ala Ser Tyr Pro His 180 185 190Phe Phe Glu Leu Lys Arg Thr Arg Thr Glu Pro Pro Gln Phe Asp 195 200 205128214PRTMycobacterium tuberculosis 128Met Pro Ser Asp Thr Ser Pro Asn Gly Leu Ser Arg Arg Glu Glu Leu1 5 10 15Leu Ala Val Ala Thr Lys Leu Phe Ala Ala Arg Gly Tyr His Gly Thr 20 25 30Arg Met Asp Asp Val Ala Asp Val Ile Gly Leu Asn Lys Ala Thr Val 35 40 45Tyr His Tyr Tyr Ala Ser Lys Ser Leu Ile Leu Phe Asp Ile Tyr Arg 50 55 60Gln Ala Ala Glu Gly Thr Leu Ala Ala Val His Asp Asp Pro Ser Trp65 70 75 80Thr Ala Arg Glu Ala Leu Tyr Gln Tyr Thr Val Arg Leu Leu Thr Ala 85 90 95Ile Ala Ser Asn Pro Glu Arg Ala Ala Val Tyr Phe Gln Glu Gln Pro 100 105 110Tyr Ile Thr Glu Trp Phe Thr Ser Glu Gln Val Ala Glu Val Arg Glu 115 120 125Lys Glu Gln Gln Val Tyr Glu His Val His Gly Leu Ile Asp Arg Gly 130 135 140Ile Ala Ser Gly Glu Phe Tyr Glu Cys Asp Ser His Val Val Ala Leu145 150 155 160Gly Tyr Ile Gly Met Thr Leu Gly Ser Tyr Arg Trp Leu Arg Pro Ser 165 170 175Gly Arg Arg Thr Ala Lys Glu Ile Ala Ala Glu Phe Ser Thr Ala Leu 180 185 190Leu Arg Gly Leu Ile Arg Asp Glu Ser Ile Arg Asn Gln Ser Pro Leu 195 200 205Gly Thr Arg Lys Glu Thr 210129224PRTMycobacterium tuberculosis 129Val Asp Glu Ile Leu Ala Arg Ala Gly Ile Phe Gln Gly Val Glu Pro1 5 10 15Ser Ala Ile Ala Ala Leu Thr Lys Gln Leu Gln Pro Val Asp Phe Pro 20 25 30Arg Gly His Thr Val Phe Ala Glu Gly Glu Pro Gly Asp Arg Leu Tyr 35 40 45Ile Ile Ile Ser Gly Lys Val Lys Ile Gly Arg Arg Ala Pro Asp Gly 50 55 60Arg Glu Asn Leu Leu Thr Ile Met Gly Pro Ser Asp Met Phe Gly Glu65 70 75 80Leu Ser Ile Phe Asp Pro Gly Pro Arg Thr Ser Ser Ala Thr Thr Ile 85 90 95Thr Glu Val Arg Ala Val Ser Met Asp Arg Asp Ala Leu Arg Ser Trp 100 105 110Ile Ala Asp Arg Pro Glu Ile Ser Glu Gln Leu Leu Arg Val Leu Ala 115 120 125Arg Arg Leu Arg Arg Thr Asn Asn Asn Leu Ala Asp Leu Ile Phe Thr 130 135 140Asp Val Pro Gly Arg Val Ala Lys Gln Leu Leu Gln Leu Ala Gln Arg145 150 155 160Phe Gly Thr Gln Glu Gly Gly Ala Leu Arg Val Thr His Asp Leu Thr 165 170 175Gln Glu Glu Ile Ala Gln Leu Val Gly Ala Ser Arg Glu Thr Val Asn 180 185 190Lys Ala Leu Ala Asp Phe Ala His Arg Gly Trp Ile Arg Leu Glu Gly 195 200 205Lys Ser Val Leu Ile Ser Asp Ser Glu Arg Leu Ala Arg Arg Ala Arg 210 215 220130236PRTMycobacterium tuberculosis 130Met Thr Thr Ser Tyr Ala Lys Ile Glu Ile Thr Gly Thr Leu Thr Val1 5 10 15Leu Thr Gly Leu Gln Ile Gly Ala Gly Asp Gly Phe Ser Ala Ile Gly 20 25 30Ala Val Asp Lys Pro Val Val Arg Asp Pro Leu Ser Arg Leu Pro Met 35 40 45Ile Pro Gly Thr Ser Leu Lys Gly Lys Val Arg Thr Leu Leu Ser Arg 50 55 60Gln Tyr Gly Ala Asp Thr Glu Thr Phe Tyr Arg Lys Pro Asn Glu Asp65 70 75 80His Ala His Ile Arg Arg Leu Phe Gly Asp Thr Glu Glu Tyr Met Thr 85 90 95Gly Arg Leu Val Phe Arg Asp Thr Lys Leu Thr Asn Lys Asp Asp Leu 100 105 110Glu Ala Arg Gly Ala Lys Thr Leu Thr Glu Val Lys Phe Glu Asn Ala 115 120 125Ile Asn Arg Val Thr Ala Lys Ala Asn Leu Arg Gln Met Glu Arg Val 130 135 140Ile Pro Gly Ser Glu Phe Ala Phe Ser Leu Val Tyr Glu Val Ser Phe145 150 155 160Gly Thr Pro Gly Glu Glu Gln Lys Ala Ser Leu Pro Ser Ser Asp Glu 165 170 175Ile Ile Glu Asp Phe Asn Ala Ile Ala Arg Gly Leu Lys Leu Leu Glu 180 185 190Leu Asp Tyr Leu Gly Gly Ser Gly Thr Arg Gly Tyr Gly Gln Val Lys 195 200 205Phe Ser Asn Leu Lys Ala Arg Ala Ala Val Gly Ala Leu Asp Gly Ser 210 215 220Leu Leu Glu Lys Leu Asn His Glu Leu Ala Ala Val225 230 235131254PRTMycobacterium tuberculosis 131Met Ser Val Asp Tyr Pro Gln Met Ala Ala Thr Arg Gly Arg Ile Glu1 5 10 15Pro Ala Pro Arg Arg Val Arg Gly Tyr Leu Gly His Val Leu Val Phe 20 25 30Asp Thr Ser Ala Ala Arg Tyr Val Trp Glu Val Pro Tyr Tyr Pro Gln 35 40 45Tyr Tyr Ile Pro Leu Ala Asp Val Arg Met Glu Phe Leu Arg Asp Glu 50 55 60Asn His Pro Gln Arg Val Gln Leu Gly Pro Ser Arg Leu His Ser Leu65 70 75 80Val Ser Ala Gly Gln Thr His Arg Ser Ala Ala Arg Val Phe Asp Val 85 90 95Asp Gly Asp Ser Pro Val Ala Gly Thr Val Arg Phe Asn Trp Asp Pro 100 105 110Leu Arg Trp Phe Glu Glu Asp Glu Pro Ile Tyr Gly His Pro Arg Asn 115 120 125Pro Tyr Gln Arg Ala Asp Ala Leu Arg Ser His Arg His Val Arg Val 130 135 140Glu Leu Asp Gly Ile Val Leu Ala Asp Thr Arg Ser Pro Val Leu Leu145 150 155 160Phe Glu Thr Gly Ile Pro Thr Arg Tyr Tyr Ile Asp Pro Ala Asp Ile 165 170 175Ala Phe Glu His Leu Glu Pro Thr Ser Thr Gln Thr Leu Cys Pro Tyr 180 185 190Lys Gly Thr Thr Ser Gly Tyr Trp Ser Val Arg Val Gly Asp Ala Val 195 200 205His Arg Asp Leu Ala Trp Thr Tyr His Tyr Pro Leu Pro Ala Val Ala 210 215 220Pro Ile Ala Gly Leu Val Ala Phe Tyr Asn Glu Lys Val Asp Leu Thr225 230 235 240Val Asp Gly Val Ala Leu Pro Arg Pro His Thr Gln Phe Ser 245 250132261PRTMycobacterium tuberculosis 132Met Gln Thr Thr Pro Gly Lys Arg Gln Arg Arg Gln Arg Gly Ser Ile1 5 10 15Asn Pro Glu Asp Ile Ile Ser Gly Ala Phe Glu Leu Ala Gln Gln Val 20 25 30Ser Ile Asp Asn Leu Ser Met Pro Leu Leu Gly Lys His Leu Gly Val 35 40 45Gly Val Thr Ser Ile Tyr Trp Tyr Phe Arg Lys Lys Asp Asp Leu Leu 50 55 60Asn Ala Met Thr Asp Arg Ala Leu Ser Lys Tyr Val Phe Ala Thr Pro65 70 75 80Tyr Ile Glu Ala Gly Asp Trp Arg Glu Thr Leu Arg Asn His Ala Arg 85 90 95Ser Met Arg Lys Thr Phe Ala Asp Asn Pro Val Leu Cys Asp Leu Ile 100 105 110Leu Ile Arg Ala Ala Leu Ser Pro Lys Thr Ala Arg Leu Gly Ala Gln 115 120 125Glu Met Glu Lys Ala Ile Ala Asn Leu Val Thr Ala Gly Leu Ser Leu 130 135 140Glu Asp Ala Phe Asp Ile Tyr Ser Ala Val Ser Val His Val Arg Gly145 150 155 160Ser Val Val Leu Asp Arg Leu Ser Arg Lys Ser Gln Ser Ala Gly Ser 165 170 175Gly Pro Ser Ala Ile Glu His Pro Val Ala Ile Asp Pro Ala Thr Thr 180 185 190Pro Leu Leu Ala His Ala Thr Gly Arg Gly His Arg Ile Gly Ala Pro 195 200 205Asp Glu Thr Asn Phe Glu Tyr Gly Leu Glu Cys Ile Leu Asp His Ala 210 215 220Gly Arg Leu Ile Glu Gln Ser Ser Lys Ala Ala Gly Glu Val Ala Val225 230 235 240Arg Arg Pro Thr Ala Thr Ala Asp Ala Pro Thr Pro Gly Ala Arg Ala 245 250 255Lys Ala Val Ala Arg 260133306PRTMycobacterium tuberculosis 133Met Thr Ile Pro Asp Ala Gln Thr Leu Met Arg Pro Ile Leu Ala Tyr1 5 10 15Leu Ala Asp Gly Gln Ala Lys Ser Ala Lys Asp Val Ile Ala Ala Met 20 25 30Ser Asp Glu Phe Gly Leu Ser Asp Asp Glu Arg Ala Gln Met Leu Pro 35 40 45Ser Gly Arg Gln Arg Thr Met Tyr Asp Arg Val His Trp Ser Leu Thr 50 55 60His Met Ser Gln Ala Gly Leu Leu Asp Arg Pro Thr Arg Gly His Val65 70 75 80Gln Val Thr Asp Thr Gly Arg Gln Val Leu Lys Ala His Pro Glu Arg 85 90 95Val Asp Met Ala Val Leu Arg Glu Phe Pro Ser Tyr Ile Ala Phe Arg 100 105 110Glu Arg Thr Lys Ala Lys Gln Pro Val Asp Ala Thr Ala Lys Arg Pro 115 120 125Ser Gly Asp Asp Val Gln Val Ser Pro Glu Asp Leu Ile Asp Ala Ala 130 135 140Leu Ala Glu Asn Arg Ala Ala Val Glu Gly Glu Ile Leu Lys Lys Ala145 150 155 160Leu Thr Leu Ser Pro Thr Gly Phe Glu Asp Leu Val Ile Arg Leu Leu 165 170 175Glu Ala Met Gly Tyr Gly Arg Ala Gly Ala Val Glu Arg Thr Ser Ala 180 185 190Ser Gly Asp Ala Gly Ile Asp Gly Ile Ile Ser Gln Asp Pro Leu Gly 195 200 205Leu Asp Arg Ile Tyr Val Gln Ala Lys Arg Tyr Ala Val Asp Gln Thr 210 215 220Ile Gly Arg Pro Lys Ile His Glu Phe Ala Gly Ala Leu Leu Gly Lys225 230 235 240Gln Gly Asp Arg Gly Val Tyr Ile Thr Thr Ser Ser Phe Ser Arg Gly 245 250 255Ala Arg Glu Glu Ala Glu Arg Ile Asn Ala Arg Ile Glu Leu Ile Asp 260 265 270Gly Ala Arg Leu Ala Glu Leu Leu Val Arg Tyr Arg Val Gly Val Gln 275 280 285Ala Val Gln Thr Val Glu Leu Leu Arg Leu Asp Glu Asp Phe Phe Asp 290 295 300Gly Leu305
Patent applications by David Roth, San Diego, CA US
Patent applications by Huaping He, San Diego, CA US
Patent applications in class Mycobacterium (e.g., Mycobacterium tuberculosis, Calmette-Guerin bacillus (i.e., BCG), etc.)
Patent applications in all subclasses Mycobacterium (e.g., Mycobacterium tuberculosis, Calmette-Guerin bacillus (i.e., BCG), etc.)