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WO2004067718A2 - Mutants de bacteries de la tuberculose depourvus d'activite necrosante - Google Patents

Mutants de bacteries de la tuberculose depourvus d'activite necrosante Download PDF

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WO2004067718A2
WO2004067718A2 PCT/US2004/002056 US2004002056W WO2004067718A2 WO 2004067718 A2 WO2004067718 A2 WO 2004067718A2 US 2004002056 W US2004002056 W US 2004002056W WO 2004067718 A2 WO2004067718 A2 WO 2004067718A2
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tuberculosis
gene
polypeptide
fragment
strain
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WO2004067718A3 (fr
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C. Harold King
William Jacobs
Daniel Okenu
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Emory University
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Emory University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium

Definitions

  • the present invention relates to novel necrosis-deficient mutants of tuberculosis bacteria and antigens identified therewith, as well as to methods for use of said mutants and antigens in the diagnosis, treatment and prevention of tuberculosis infection.
  • Tuberculosis caused in humans by Mycobacterium tuberculosis, continues to be a major cause of morbidity and death throughout the world. Tuberculosis is reported to be the most frequent cause of death from a single infectious agent in persons from 15-49 years of age, and results in 1.5 to 3 million deaths each year. It is estimated that as many as one- third of the world's population has been infected with the organism that causes tuberculosis. However, many of the individuals infected with M. tuberculosis do not develop clinical disease. The host's immune response is usually adequate to control the primary infection but is virtually never capable of eradicating the organism. In 5-10% of those individuals with latent infections, the infections will reactivate and result in the development of active tuberculosis.
  • tuberculosis The natural history of tuberculosis and the interaction of M. tuberculosis with its host is complex, and entails distinct phases of exposure, infection, bacterial replication and dissemination, the host's immune response that usually controls the primary infection or leads to the active disease, development and maintenance of latency, and subsequent reactivation of latent infection and development of active disease of latently infected individuals.
  • the development of improved control strategies for tuberculosis will require a better understanding of M. tuberculosis pathogenesis. Before this can be accomplished, several important questions on the pathophysiology of tuberculosis remain: i.) what are the early events in the pathogenesis of tuberculosis; specifically how M.
  • tuberculosis infects, survives, multiplies, and interacts with the different host cells found in the human lung alveolus, ii.) what are the determinants for the dissemination of M. tuberculosis from the alveoli leading to hematogenous spread in the lungs, iii) what are the mechanisms involved in the pathology of the lung, iv.) do bacterial factors also contribute directly or indirectly to tissue destruction and cavitary disease, and v.) what are the mechanisms for reactivation from the latent state of infection to active disease.
  • M. tuberculosis has clearly evolved differently from other intracellular pathogens such as Listeria monocytogenes and Salmonella typhimurium that, unlike M. tuberculosis, must survive in the environment outside of its host.
  • M. tuberculosis infection of host cells is often similar to infection by these other mtracellular pathogens, suggesting that many of the known virulence determinants described for other intracellular pathogens may have been modified differently in M.
  • M. tuberculosis through adaptation to host specificity.
  • M. tuberculosis clearly invades epithelial cells, directs the intracellular trafficking of its phagosomes to avoid killing upon infection of host cells, and causes apoptosis and necrosis in tissues, but only a small number of significantly homologous virulence determinants found in other intracellular bacterial pathogens are present in the genome of M. tuberculosis. Modification of known virulence determinants in the genome of M. tuberculosis is supported by a recent study. M.
  • tuberculosis was found to possess a gene with only 38% homology to the Salmonella mgtC gene, a known virulence factor of Salmonella required for the ability to acquire intra-phagosomal magnesium and for growth within macrophages.
  • Salmonella mgtC gene a known virulence factor of Salmonella required for the ability to acquire intra-phagosomal magnesium and for growth within macrophages.
  • mutation of this gene in M. tuberculosis attenuated its growth in human macrophages and in the lungs and spleens of mice.
  • Tuberculosis is a necrotic disease that is primarily spread though the air by inhalation of small droplet nuclei containing M.
  • tuberculosis from the damaged lungs of diseased individuals into the lungs of a susceptible host.
  • a hallmark of tuberculosis is the development of caseating necrosis, parenchymal lung destruction, and cavity formation.
  • an advanced stage of tissue destruction develops until a cavity in the tissues is formed called a tubercle.
  • Advanced pulmonary tuberculosis is then caused by the locally destructive process of multiple cavitary lesions, leading to disruption of the bronchial epithelium and release of the bacilli outside the lungs. Formation of these cavities and/or destruction of the bronchial epithelium therefore must also play an important role in the transmission of disease to the next host.
  • BCG Bacillus Calmette-Guerin
  • M. bovis an avirulent strain of M. bovis.
  • BCG Bacillus Calmette-Guerin
  • diagnosis of tuberculosis is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative).
  • Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals. Thus, there is currently no effective, safe, inexpensive vaccination against tuberculosis. Further, there is no rapid, inexpensive point-of-care diagnostic test for tuberculosis. The standard of care for pharmaceutical treatment of the disease consists of administering multiple antibiotics over an extended period, generally several months. In other words, prevention, diagnosis, and treatments for tuberculosis are all lacking. We believe that the invention described herein could have significant impact in all three areas, and could be useful for development of a vaccine, diagnostic, or pharmaceutical entity.
  • the present invention provides in part novel necrosis-deficient tuberculosis bacterial strains and microbiological cultures thereof.
  • the strain of tuberculosis bacteria is a member of the Mycobacterium tuberculosis complex.
  • the strain is a M. tuberculosis strain that may comprise a mutation in at least one gene involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • said gene is located within the RD1 chromosomal region.
  • such gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • a necrosis-deficient M. tuberculosis strain may comprise a mutation in at least one gene involved in tuberculosis-induced epithelial cell permeation and or necrosis, wherein said M. tuberculosis strain maintains intracellular and extracellular growth rates comparable to a parent strain.
  • a strain may not induce cell death of human lung epithelial cells in vitro, but still maintain intracellular and extracellular growth rates comparable to a parent strain.
  • a strain may be significantly attenuated in virulence in an aerosol infection SCID mouse model of tuberculosis, but still maintain intracellular and extracellular growth rates comparable to a parent strain.
  • polypeptides and fragments thereof that are encoded by genes involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • Such polypeptides may in certain embodiments comprise M. tuberculosis antigens, and may be identified, for example, using the necrosis-deficient mutants described above.
  • Recombinant versions of such polypeptides, as well as fusions, domains, fragments, variants and derivatives thereof, are also provided by the present invention.
  • the present invention provides an isolated recombinant polypeptide or fragment thereof having, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding polypeptide or fragment thereof encoded by a gene from M. tuberculosis involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • a gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • the isolated recombinant polypeptide or fragment thereof is encoded by all or a portion of SEQ ID NO: 1. Such polypeptides may be optionally tagged, for example, with a 6x His tag.
  • the present invention also provides isolated nucleic acid sequences encoding the polypeptides or polypeptide fragments, as well as vectors, host cells, and cultures thereof.
  • the polypeptides and fragments of the invention are antigens. Still further, the present invention provides antibodies specific to such antigens. In certain embodiments, the antibodies are specific for an antigenic polypeptide or antigenically active fragment thereof encoded by a gene selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877. In certain embodiments, an antibody may be specific for a Rv3351c polypeptide or fragment thereof. In other embodiments, an antibody may be specific for the polypeptide encoded by SEQ ID NO: 1, or an antigenically active fragment of said polypeptide.
  • necrosis-deficient tuberculosis bacterial strains may comprise immunogenic compositions and vaccines.
  • a strain of the invention is attenuated in virulence and/or retains its immunogenic properties without being able to cause a tuberculosis infection.
  • a polypeptide of the present invention may correspond to a protein that is essential for virulence or infectivity of M. tuberculosis.
  • such strains, polypeptides, and nucleic acids encoding such polypeptides may be used as part of an immunogenic composition or vaccine, for example, formulated in a pharmaceutically acceptable carrier, to prevent tuberculosis infection.
  • immunogenic compositions or vaccines may further comprise other known tuberculosis vaccines, such as, for example, Bacillus Calmette-Guerin, or comprise other compounds such as adjuvants, or combinations of the strains and antigens of the invention.
  • the immunogenic compositions and vaccines of the present invention may be used in methods of treating and preventing tuberculosis in mammals, for example, humans.
  • a method for eliciting an immunogenic response in a mammal may comprise administering to a mammal an effective amount of aM. tuberculosis strain of the invention.
  • a method for eliciting an immunogenic response in a mammal may comprise administering to an subject at least one polypeptide or antigenically active fragment thereof having for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding polypeptide or fragment thereof encoded by a gene from M. tuberculosis involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • such gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • a method for eliciting an immunogenic response in a mammal may comprise administering to an subject a nucleic acid encoding at least one antigenically active gene product, for example a polypeptide or antigenically active fragment thereof, having for example, at least about 80%, at least about 85%>, at least about 90%>, at least about 95%), at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding gene product encoded by a gene from M. tuberculosis.
  • the nucleic acid sequence may be comprised of DNA or RNA, and may optionally comprise a vector.
  • the gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • a method for vaccinating a mammal against tuberculosis may comprise in certain embodiments administering to an subject at least one polypeptide or antigenically active fragment thereof having for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%>, at least about 98%>, or at least about 99% identity to the corresponding polypeptide or fragment thereof encoded by a gene from, for example, M. tuberculosis involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • a method for vaccinating a mammal against tuberculosis comprises administering to an subject a nucleic acid encoding at least one antigenically active gene product, for example a polypeptide or antigenically active fragment thereof, having for example, at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 96%o, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding gene product encoded by a gene from, for example, M.
  • at least one antigenically active gene product for example a polypeptide or antigenically active fragment thereof, having for example, at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 96%o, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding gene product encoded by a gene from, for example, M.
  • the nucleic acid sequence may be comprised of DNA or RNA, and may optionally comprise a vector.
  • the gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • a method may comprise: detecting in a sample the presence of at least one polypeptide encoded by a M. tuberculosis gene selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877. Detection may be accomplished, for example, by an antibody specific for the polypeptide to be detected.
  • test compounds may be screened and thereby evaluated for ability to normalize the level of expression of a gene involved in epithelial cell permeation or necrosis, for example, Rv3351c, Rv3875 or Rv3874.
  • test compounds may be screened and thereby evaluated for ability to modulate the activity of a protein encoded by a gene involved in epithelial cell permeation or necrosis, for example, Rv3351c.
  • Assays and methods of developing assays appropriate for use in the methods described above are known to those of skill in the art and, as will be appreciated by those skilled in the art, may be used as suitable with the methods of the present invention. Further, the present invention provides methods of treating tuberculosis using pharmaceutical compositions comprised of therapeutic agents identified using the screening methods provided by the invention.
  • kits including the subject bacterial strains, nucleic acids, polypeptides, antibodies, and other subject materials, and optionally instructions for their use. Kits comprising the pharmaceutical compositions of the present invention are also within the scope of the invention. Uses for such kits include, for example, diagnostic and therapeutic applications.
  • FIGURES Figure 1 depicts representative 96-well plate results for the % A549 cell permeation screening assay showing 64 hygromycin resistant (black bars) and eight wild type M. tuberculosis (white bars) strain Erdman transfers after 48 infection of A549 cells. Recombinants ranged from ⁇ 1%> to 17%> cell permeation of A549 cells after 48 h incubation.
  • Figure 2A depicts representative 96-well plate results for the % A549 cell permeation screening assay showing four (six replicate each) hygromycin resistant clones and wild type M. tuberculosis (eight replicate) strain Erdman transfers after 48 h incubation.
  • Figure 2B depicts representative 96-well plate results for the % A549 cell permeation screening assay showing four (six replicate) hygromycin resistant clones and wild type M. tuberculosis (eight replicate) strain Erdman transfers after 72 h incubation.
  • Figure 3 depicts growth curves of M. tuberculosis recombinants and the wild type Erdman. Equal volumes of inoculum were added to duplicate tubes containing 5 ml of 7H9/TP medium and O.D.goo was measured in triplicate at each indicated time point. Evidence that inoculum size was similar is shown at 24 h post inoculation.
  • Figure 4 depicts CFU obtained from intracellular growth of wild type and mutant M. tuberculosis within A549 cells. Although there was a significant difference in CFU after 24 h post infection, there was no significant difference (P>0.05) after this time point during the infection when LDH release and necrosis were evident (day three post infection). Data shown are means from two different experiments of triplicate monolayers Error bars, standard errors.
  • Figure 5 depicts LDH release from lung epithelial cells at 72 h post-infection is reduced in the necA mutant and is restored back to the wild type by complementation with the Rv3874/Rv3875 operon. Note that the necA mutant is less attenuated for permeation of epithelial cells ( ⁇ 65%> of wild type) than the necB mutant ( ⁇ 10%> of wild type; Figure 6).
  • Figure 6 depicts LDH release from lung epithelial cells at 48 h post-infection is reduced in the nec-9 mutant and is restored back to the wild type by complementation with Rv3351c.
  • FIG. 8 The NecA gene is required for virulence in SCID mice.
  • Ten SCID mice were infected intravenously with 2 x 10 6 M. tuberculosis Erdman plus pMV306 an vector control (squares), Rv3874::TN5370 mutant plus pMV306 K n vector control (triangles), and
  • Figure 9 illustrates generation of mutants in the RDl region of M. tuberculosis and M. bovis.
  • Figure 9A is a schematic of M. tuberculosis H37Rv RDl region showing predicted Ncol sites. Arrows at the top represent the genes in this region. UFSs and DFSs used to generate the knockout are indicated as filled bars above the grid line. Each increment in the grid line represents 1 kbp.
  • the RDl sequence deleted from M. bovis BCG is represented by an open bar spanning from Rv3871 to Rv3879c. The site of the insertion of transposon Tn5370 is also indicated.
  • Figure 9B depicts a Southern analysis of the Ncol- digested genomic DNA isolated from the wild type and the ⁇ RD1 mutants generated by using specialized transduction in M. tuberculosis and M. bovis.
  • the probe used in the Southern analysis was either DFS (Left), demonstrating the deletion of RDl, or IS6110-specif ⁇ c (Right). The IS6110 probe is used to characterize the four different strains.
  • Figure 10 illustrates deletion of RDl in tuberculosis conferring an attenuation of virulence in vivo.
  • Figure 10A depicts survival experiment using SCED mice i.v. infected with 2 X 106 cfu per mouse. Infection was carried out as described in Example 5. Strains used were M. tuberculosis H37Rv (•), M. tuberculosis ⁇ RD1 (_), M. tuberculosis ⁇ RD1 (pYUB412::Rv3860-Rv3885c) ( E), and M. bovis BCG (,).
  • Figure 10B depicts survival experiment with BALB c mice i.v. infected with M.
  • FIG. 10C depicts growth kinetics in the lungs of BALB_c mice i.v. infected with M. bovis BCG ( ⁇ ), M. tuberculosis H37Rv (•), and M. tuberculosis H37Rv ⁇ RD1 ( ⁇ ).
  • the infecting dose per mouse was 2 X 106 cfu.
  • Data represent the mean of cfus from three mice per time point.
  • Figure 11 contains a Table comparing vaccination results obtained using M. tuberculosis H37rv andM bovis BCG.
  • Figure 12 illustrates results indicating that the cfp-10 esat-6 (Rv3874_Rv3875) operon of RDl is required for host cell lysis.
  • Figure 12A depicts a LDH release assay of infected lung epithelial cells, at 72 h postinfection. Cells were infected at a multiplicity of infection of 10:1, and supernatants were analyzed for LDH release, wt, M.
  • tuberculosis Erdman _ pYUB412 empty vector
  • mt mc24513 _ cfplO::Tn5370 _ pYUB412
  • c mc24513 _ ⁇ YUB412::Rv3860-Rv3885c
  • DRDl M. tuberculosis Erdman _RD1.
  • Values are means _ SD of triplicate measurements.
  • Figure 12B depicts a LDH release assay of bone marrow-derived macrophages at day 7 postinfection. Labels wt, mt, and c are as in A. Values are means _ SD of triplicate measurements.
  • Figure 12C depicts a necrosis determining assay, measuring histone-associated DNA fragments in the supernatant. Labels wt, mt, and c are as in A and B.
  • Figure 12D shows the M. tuberculosis necrosis phenotype is inhibited by 5 mM exogenous glycine (_g), compared with the control, in which there is no exogenous glycine (g). Values are means _ SD of triplicate measurements.
  • Figure 12E shows that ESAT-6, but not CFP-10, is sufficient to induce disruption, leading to total destruction (see arrowhead) of an artificial lipid bilayer. Artificial planal bilayers were constructed by using the lipid diphytanoylphosphatidylcholine.
  • Figure 13 shows that the loss of cfp-10_esat-6 (Rv3874_Rv3875) confers attenuation of virulence.
  • Figure 13A depicts survival in SCID mice i.v. infected with 2 _ 106 cfu of M. tuberculosis Erdman _ ⁇ YUB412 (wt), mc24513 _ cfplO::Tn5370 _ pYUB412 (mt), and mc24513 _ pYUB412::Rv3860-Rv3885c (c).
  • Figure 13B depicts electron micrographs of early lesions after high-dose aerosol infection of BALB_c mice with M.
  • Figure 13 Bl depicts mutant-infected macrophage in the alveolar airspace. This result was not observed in the wild-typeinfected mice at this time point.
  • Figure 13 B2 depicts macrophage in the lumen of the airspace with large numbers of intracellular mutant bacilli.
  • Figure 13 B3 depicts wild-type bacillus residing within a macrophage, located interstitially. This result was not observed in mutant-infected mice at this time point.
  • Figure 13 B4 depicts a cell within the alveolar space with ingested wild- type bacillus is lysed, resulting in the release of the organism. This result was not observed in mice infected with the mutant bacilli, as, alveolar space; bv, blood vessel; lc, lytic cell; m, macrophage; p, type I pneumocyte. Arrows denote mycobacteria.
  • Figure 14 shows that the genes Rv3871 and Rv3876_Rv3877 of RDl are required for virulence and for the secretion of ESAT-6.
  • Figure 14A depicts the survival time of SCID mice infected i.v. with 2 _ 106 cfu of M. tuberculosis H37Rv (J, _Rv3871 (F), _Rv3872_3 (CE),_Rv3874_5 Q, and_Rv3876_7 ( ⁇ ).
  • Figure 14B depicts Western analyses of the whole cell extracts and culture filtrates. Arrow indicates the ESAT-6-specific band.
  • Figure 14 Bl depicts culture filtrates probed with both anti-ESAT-6 primary antibody HYB76-8 and goat anti-mouse Ig secondary antibody.
  • Figure 14 B2 depicts culture filtrates probed only with secondary antibody.
  • Figure 14 B3 depicts whole-cell lysates probed with both primary and secondary antibodies.
  • lanes are as follows: lane 1, purified ESAT-6 protein (50 ng); lane 2, _Rv3871; lane 3, _Rv3872_3; lane 4, _Rv3874_5; and lane 5, _Rv3876_7.
  • Figure 14 B4 depicts Western analysis of culture filtrates of M.
  • tuberculosis H37Rv, H37Rv _RD1, and complemented strain H37Rv _RD1 (pYUB412::Rv3860-Rv3885c), reacted with both primary and secondary antibodies.
  • Lane 1 purified ESAT-6 protein (50 ng); lane 2, H37Rv; lane 3, H37Rv _RD1; and lane 4, H37Rv _RD1 (pYUB412::Rv3860-Rv3885c).
  • Figure 15 depicts expression of MTB Rv3351c gene in the pET29a(+) vector.
  • Expression construct was transformed into BL21(DE3) E. coli cells, induced at OD600 of 0.6 with a final JPTG concentration of lmM for 3 h at 37°C.
  • Gene product was IgG positive against confirmed TB patients sera samples.
  • Figure 16 depicts Western blotting of M. tuberculosis Rv3351c recombinant protein (30 kDa) against a confirmed TB serum sample.
  • an element means one element or more than one element.
  • an “adjuvant” is a substance that in combination with specific antigen may produce a greater immunogenic response than the antigen alone.
  • an “antigen” is a substance that stimulates the production or mobilization of antibodies.
  • An antigen may be, for example, a foreign protein, toxin, bacterium, or other substance.
  • the term “antigenically active” also refers to a substance which has the ability to act as an antigen. In particular, as used herein, it refers to a substance which is a fragment, derivative, or variant of a particular antigen, but still retains the antigenic properties of the antigen.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with a polypeptide of the invention. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as is suitable for whole antibodies. For example, F(ab') 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab') 2 fragment can be treated to reduce disulf ⁇ de bridges to produce Fab' fragments.
  • the antibody of the present invention is further intended to include bispecific and chimeric molecules, as well as single chain (scFv) antibodies. Also within the scope of the invention are trimeric antibodies, humanized antibodies, human antibodies, and single chain antibodies.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • binding refers to an association, which may be a stable association, between two molecules, e.g., between a polypeptide of the invention and a binding partner, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • a "fusion protein” or “fusion polypeptide” refers to a chimeric protein as that term is known in the art and may be constructed using methods known in the art. In many examples of fusion proteins, there are two different polypeptide sequences, and in certain cases, there may be more. The sequences may be linked in frame.
  • a fusion protein may include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergenic”, etc. fusion expressed by different kinds of organisms.
  • the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide. In the case where more than one amino acid sequence is fused to a first polypeptide, the fusion sequences may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences.
  • the fusion polypeptides may be fused to the N-terminus, the C-terminus, or the N- and C-terminus of the first polypeptide.
  • Exemplary fusion proteins include polypeptides comprising a glutathione S-transferase tag (GST-tag), histidine tag (His-tag), an immunoglobulin domain or an immunoglobulin binding domain.
  • gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide having exon sequences and optionally intron sequences.
  • intron refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.
  • a "gene involved in tuberculosis-induced host cell permeation and/or necrosis” refers to a gene which enables a tuberculosis bacteria to engage in host epithelial cell membrane permeation and/or induce necrosis such cells cells.
  • immunogenic refers to the ability of a substance to elicit an immune response.
  • An "immunogenic composition” or “immunogenic substance” is a composition or substance which elicts an immune response.
  • An “immune reponse” refers to the reaction of a subject to the presence of an antigen, which may include at least one of the following: making antibodies, developing immunity, developing hypersensitivity to the antigen, and developing tolerance.
  • immunogen refers to the ability of an organism to resist or overcome an infection.
  • protection immunity refers to the ability of an organism to resist an infection.
  • isolated polypeptide refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • isolated nucleic acid refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which (1) is not associated with the cell in which the "isolated nucleic acid” is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
  • mammal is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).
  • modulation when used in reference to a functional property or biological activity or process (e.g., enzyme activity or receptor binding), refers to the capacity to either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a quality of such property, activity or process. In certain instances, such regulation may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
  • modulator refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species or the like (naturally-occurring or non-naturally-occurring), or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, that may be capable of causing modulation.
  • Modulators may be evaluated for potential activity as inhibitors or activators (directly or indirectly) of a functional property, biological activity or process, or combination of them, (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, anti-microbial agents, inhibitors of microbial infection or proliferation, and the like) by inclusion in assays, hi such assays, many modulators may be screened at one time.
  • the activity of a modulator may be known, unknown or partially known.
  • Mycobacterium tuberculosis is abbreviated as “M. tuberculosis” herein.
  • the "Mycobacterium tuberculosis complex" of mycobacteria includes M. tuberculosis, M. bovis, M. avium, M. africanum, M. microti, and atypical mycobacteria.
  • Mycobacterium tuberculosis complex bacteria are the causative agents of tuberculosis. Such bacteria are also referred to herein as “tuberculosis bacteria.”
  • a “nee gene” refers to a gene of a tuberculosis strain that is involved in host epithelial cell membrane permeation and/or induction of cell necrosis.
  • nucleic acid refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • nucleic acid of the invention refers to a nucleic acid encoding a polypeptide of the invention.
  • operably linked when describing the relationship between two nucleic acid regions, refers to a juxtaposition wherein the regions are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules (e.g., inducers and polymerases) are bound to the control or regulatory sequence(s).
  • a "patient,” “subject” or “host” to be treated by the subject method may mean either a human or non-human animal.
  • compositions and dosages thereof within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body.
  • pharmaceutically acceptable carrier refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof.
  • materials which may serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • phenotype refers to the entire physical, biochemical, and physiological makeup of a cell, e.g., having any one trait or any group of traits.
  • polypeptide and the terms “protein” and “peptide” which are used interchangeably herein, refers to a polymer of amino acids.
  • exemplary polypeptides include gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
  • polypeptide fragment when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.
  • Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 or more amino acids long.
  • a fragment can retain one or more of the biological activities of the reference polypeptide.
  • a fragment may comprise a druggable region, and optionally additional amino acids on one or both sides of the druggable region, which additional amino acids may number from 5, 10, 15, 20, 30, 40, 50, or up to 100 or more residues.
  • fragments can include a sub-fragment of a specific region, which sub-fragment retains a function of the region from which it is derived.
  • a fragment may have immunogenic properties.
  • purified refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a “purified fraction” is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present.
  • the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species (including the one of interest) dissolved or dispersed are taken into account.
  • a purified composition will have one species that comprises more than about 80 percent of all species present in the composition, more than about 85%, 90%>, 95%, 99% or more of all species present.
  • the object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.
  • a skilled artisan may purify a polypeptide of the invention using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide may be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis, mass- spectrometry analysis and the methods described in the Exemplification section herein.
  • recombinant protein or “recombinant polypeptide” refer to a polypeptide which is produced by recombinant DNA techniques.
  • An example of such techniques includes the case when DNA encoding the expressed protein is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the protein or polypeptide encoded by the DNA.
  • regulatory sequence is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators and promoters, that are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operably linked.
  • regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990), and include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the tip system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • control sequences may differ depending upon the host organism.
  • such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences.
  • the term "regulatory sequence" is intended to include, at a minimum, components whose presence may ' influence expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • transcription of a polynucleotide sequence is under the control of a promoter sequence (or other regulatory sequence) which controls the expression of the polynucleotide in a cell-type in which expression is intended. It will also be understood that the polynucleotide can be under the control of regulatory sequences which are the same or different from those sequences which control expression of the naturally-occurring form of the polynucleotide.
  • Rv3351c refers to the NecB gene
  • An exemplary NecB gene sequence is SEQ ID NO: 1: gtgctggcgagctgcccggcgcggtccggcgcggctgtcgcggatgcgatcaagtccgcggttggagtgcaacccagtggagtt gagcacaagacgctgcgccgtatggacctggtgaggtatctggccggcggccatacgacctatccgccggagggcttcgtggct ggatccgatgtcatcgggacgacgaatccggccgcggcccaagccatcgtcgtcgcgccatcggaacatggccacccgctgcggg ccgcgcgcgccatcggaacatggccacccgctgcggg cg
  • NecB gene sequences also may be obtained GenBank Accession Numbers NC_002945; NC_000962; BX842582; and BX248345.
  • the term Rv3351c also includes portions of, homologs of, orthologs of, variants of, isoforms of, allelic variants or other sequences at least about 80% identical to any NecB gene.
  • sequence homology refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences.
  • sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence from a desired sequence (e.g., SEQ. ID NO: 1) that is compared to some other sequence.
  • Gaps in either of the two sequences are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are used more frequently, with 2 bases or less used even more frequently.
  • sequence identity means that sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for polypeptides) over a window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical amino acids occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Methods to calculate sequence identity are known to those of skill in the art and described in further detail below.
  • small molecule refers to a compound, which has a molecular weight of less than about 5 kD, less than about 2.5 kD, less than about 1.5 kD, or less than about 0.9 kD.
  • Small molecules may be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention.
  • small organic molecule refers to a small molecule that is often identified as being an organic or medicinal compound, and does not include molecules that are exclusively nucleic acids, peptides or polypeptides.
  • soluble as used herein with reference to a polypeptide of the invention or other protein, means that upon expression in cell culture, at least some portion of the polypeptide or protein expressed remains in the cytoplasmic fraction of the cell and does not fractionate with the cellular debris upon lysis and centrifugation of the lysate. Solubility of a polypeptide may be increased by a variety of art recognized methods, including fusion to a heterologous amino acid sequence, deletion of amino acid residues, amino acid substitution (e.g., enriching the sequence with amino acid residues having hydrophilic side chains), and chemical modification (e.g., addition of hydrophilic groups).
  • solubility of polypeptides may be measured using a variety of art recognized techniques, including, dynamic light scattering to determine aggregation state, UN absorption, centrifugation to separate aggregated from non-aggregated material, and SDS gel electrophoresis (e.g., the amount of protein in the soluble fraction is compared to the amount of protein in the soluble and insoluble fractions combined).
  • the polypeptides of the invention When expressed in a host cell, the polypeptides of the invention may be at least about 1%, 2%, 5%, 10%>, 20%, 30%, 40%), 50%), 60%>, 70%>, 80%, 90% or more soluble, e.g., at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total amount of protein expressed in the cell is found in the cytoplasmic fraction.
  • a one liter culture of cells expressing a polypeptide of the invention will produce at least about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 milligrams or more of soluble protein.
  • a polypeptide of the invention is at least about 10% soluble and will produce at least about 1 milligram of protein from a one liter cell culture.
  • polynucleotides, oligonucleotides and nucleic acids of the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • Stringent conditions may be used to achieve selective hybridization conditions as known in the art and discussed herein.
  • the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and nucleic acids of the invention and a nucleic acid sequence of interest will be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or more.
  • hybridization and washing conditions are performed under stringent conditions according to conventional hybridization procedures and as described further herein.
  • strain refers to a tuberculosis bacterial cell derived from a primary culture or cell line by the selection and cloning of cells having specific properties.
  • stringent conditions or “stringent hybridization conditions” refer to conditions which promote specific hydribization between two complementary polynucleotide strands so as to form a duplex. Stringent conditions may be selected to be about 5°C lower than the thermal melting point (Tm) for a given polynucleotide duplex at a defined ionic strength and pH. The length of the complementary polynucleotide strands and their GC content will determine the Tm of the duplex, and thus the hybridization conditions necessary for obtaining a desired specificity of hybridization.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the a polynucleotide sequence hybridizes to a perfectly matched complementary strand. In certain cases it may be desirable to increase the stringency of the hybridization conditions to be about equal to the Tm for a particular duplex.
  • Tm Tm-C base pairs in a duplex are estimated to contribute about 3°C to the Tm, while A-T base pairs are estimated to contribute about 2°C, up to a theoretical maximum of about 80-100°C.
  • G-C stacking interactions, solvent effects, the desired assay temperature and the like are taken into account.
  • Hybridization may be carried out in 5xSSC, 4xSSC, 3xSSC, 2xSSC, lxSSC or 0.2xSSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24 hours.
  • the temperature of the hybridization may be increased to adjust the stringency of the reaction, for example, from about 25oC (room temperature), to about 45oC, 50oC, 55oC, 60oC, or 65oC.
  • the hybridization reaction may also include another agent affecting the stringency, for example, hybridization conducted in the presence of 50% formamide increases the stringency of hybridization at a defined temperature.
  • the hybridization reaction may be followed by a single wash step, or two or more wash steps, which may be at the same or a different salinity and temperature.
  • the temperature of the wash may be increased to adjust the stringency from about 25oC (room temperature), to about 45oC, 50oC, 55oC, 60oC, 65oC, or higher.
  • the wash step may be conducted in the presence of a detergent, e.g., 0.1 or 0.2% SDS.
  • a detergent e.g., 0.1 or 0.2% SDS.
  • hybridization may be followed by two wash steps at 65oC each for about 20 minutes in 2xSSC, 0.1% SDS, and optionally two additional wash steps at 65oC each for about 20 minutes in 0.2xSSC, 0.1%SDS.
  • Exemplary stringent hybridization conditions include overnight hybridization at 65oC in a solution comprising, or consisting of, 50% formamide, lOxDenhardt (0.2% Ficoll, 0.2%) Polyvinylpyrrolidone, 0.2%> bovine serum albumin) and 200 tig/ml of denatured carrier DNA, e.g., sheared salmon sperm DNA, followed by two wash steps at 65oC each for about 20 minutes in 2xSSC, 0.1% SDS, and two wash steps at 65oC each for about 20 minutes in 0.2xSSC, 0.1%SDS.
  • denatured carrier DNA e.g., sheared salmon sperm DNA
  • Hybridization may consist of hybridizing two nucleic acids in solution, or a nucleic acid in solution to a nucleic acid attached to a solid support, e.g., a filter.
  • a prehybridization step may be conducted prior to hybridization. Prehybridization may be carried out for at least about 1 hour, 3 hours or 10 hours in the same solution and at the same temperature as the hybridization solution (without the complementary polynucleotide strand).
  • substantially identical means that two protein sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, typically share at least about 70 percent sequence identity, alternatively at least about 80, 85, 90, 95 percent sequence identity or more. In certain instances, residue 004/067718
  • systemic administration means the administration of a subject supplement, composition, therapeutic or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • test compound refers to a molecule to be tested by one or more screening method(s) as a putative modulator of a polypeptide of the invention or other biological entity or process.
  • a test compound is usually not known to bind to a target of interest.
  • control test compound refers to a compound known to bind to the target (e.g., a known agonist, antagonist, partial agonist or inverse agonist).
  • test compound does not include a chemical added as a control condition that alters the function of the target to determine signal specificity in an assay.
  • control chemicals or conditions include chemicals that 1) nonspecifically or substantially disrupt protein structure (e.g., denaturing agents (e.g., urea or guanidinium), chaotropic agents, sulfhydryl reagents (e.g., dithiothreitol and b-mercaptoethanol), and proteases), 2) generally inhibit cell metabolism (e.g., mitochondrial uncouplers) and 3) non-specifically disrupt electrostatic or hydrophobic interactions of a protein (e.g., high salt concentrations, or detergents at concentrations sufficient to non-specifically disrupt hydrophobic interactions).
  • test compound also does not include compounds known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject.
  • test compounds include, but are not limited to, peptides, nucleic acids, carbohydrates, and small molecules.
  • the term "novel test compound” refers to a test compound that is not in existence as of the filing date of this application.
  • the novel test compounds comprise at least about 50%>, 75%>, 85%, 90%, 95% or more of the test compounds used in the assay or in any particular trial of the assay.
  • therapeutically effective amount refers to that amount of a modulator, drug or other molecule which is sufficient to effect treatment when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • treating as used herein is intended to encompass curing as well as ameliorating at least one symptom of any condition or disease.
  • the term “tuberculosis” refers to any disease or disorder caused by an infection with a member of the Mycobacterium tuberculosis complex.
  • vaccine refers to a substance that elicits an immune response and also confers protective immunity upon a subject.
  • vector refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked.
  • One type of vector which may be used in accord with the invention is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of
  • Plasmids which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome.
  • vector refers to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome.
  • plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • the present invention provides in part novel necrosis-deficient tuberculosis bacteria strains and microbiological cultures thereof.
  • the strain of tuberculosis bacteria is a member of the Mycobacterium tuberculosis complex.
  • Such strains may comprise, for example, a mutation in at least one gene involved in tuberculosis- induced epithelial cell permeation and/or necrosis.
  • Mutants of tuberculosis bacteria with reduced membrane permeation ability may be identified using any mutagenesis method, for example random mutagenesis.
  • random transposon mutagenesis may be used to generate a library of mutants.
  • Example 2 further describes the introduction of a transposon into the permeating/necrosis positive wild type strain Erdman. Such mutants may be screened, for example, for reduction in LDH release upon cellular infection, using wild type bacteria as a control, and then determine if these LDH release mutants are also defective in the ability to induce necrosis.
  • transposon disrupted gene is the gene responsible for the loss of the necrosis phenotype may be identified as the transposon disrupted gene, for example, as described in Examples 4 and 5.
  • an integrating vector such as for example, ⁇ MV306 kan
  • ⁇ MV306 kan may be used to restore the functional gene in the transposon-disrupted strains. This will restore the parental phenotype to the mutant strain.
  • non-revertible mutants may be constructed using homologous recombination. These stable mutants and wild type M. tuberculosis may then be studied in a mouse model of tuberculosis to determine the role of host cell necrosis to the growth, tissue pathology, hematogenous spread, and virulence in mice.
  • the strain is a M. tuberculosis strain that may comprise a mutation in at least one gene involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • said gene is located within the RDl chromosomal region.
  • such gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • such mutation may result in substantial disablement of said gene.
  • the mutation may be in one gene, for example, the Rv3351c gene, and in certain embodiments may result in substantial disablement of that gene.
  • the strain comprises the TPC 2.6.2E mutant (alternatively called Rv3351::TN5370 or necB). In other embodiments, the strain comprises the TPC2.9.3G mutant (alternatively called Rv3874::TN5370 or necA).
  • TPC2.9.3G mutant alternatively called Rv3874::TN5370 or necA.
  • tuberculosis Erdman may be further screened and analyzed for selection of additional mutants with defects in membrane permeation and necrosis of epithelial cells relevant to virulence in the SCID model as described above.
  • mutants that appear virulent in the intravenous infection of SCID mice may be tested by the aerosol infection SCID mouse model to determine if these mutants warrant further study using the methods described above.
  • a hygromycin-tagged Mariner transposon phage library may be screened for mutants defective in necrosis as described above.
  • Selected mutants and complemented clones may be studied further in both in vitro and in vivo models.
  • each mutant pair and nee gene product may be studied based on a selection criteria of which nee genes are directly involved in virulence and pathology in the SCID and Balb/c mouse models, as described in Example 8.
  • a necrosis-deficient M. tuberculosis strain may comprise a mutation in at least one gene involved in tuberculosis-induced epithelial cell permeation and/or necrosis, wherein said M. tuberculosis strain maintains intracellular and extracellular growth rates comparable to a parent strain.
  • a strain may not induce cell death of human lung epithelial cells in vitro, but still maintain intracellular and extracellular growth rates comparable to a parent strain.
  • a strain may be significantly attenuated in virulence in an aerosol infection SCID mouse model of 004/067718
  • tuberculosis but still maintain intracellular and extracellular growth rates comparable to a parent strain.
  • polypeptides encoded by genes involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • Such polypeptides may comprise antigens, as described below, and may be identified, for example, using the necrosis-deficient mutant strains described above.
  • Recombinant versions of such polypeptides, as well as fusions, domains, fragments, variants and derivatives thereof, are also provided by the present invention.
  • the present invention provides an isolated recombinant polypeptide or fragment thereof having, for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding polypeptide or fragment thereof encoded by a gene from M. tuberculosis involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • a gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • such gene exhibits at least about 80%, at least about 85%, at least about 90%, at least about 95%,, at least about 96%, at least about 97%o, at least about 98%, or at least about 99% identity to Rv3351c.
  • the isolated recombinant polypeptide or fragment thereof is encoded by all or a portion of SEQ ID NO: 1. Such polypeptides may be optionally tagged, for example, with a 6x His tag.
  • the polypeptides of the invention may be modified so as to increase their immunogenicity.
  • a polypeptide such as an antigenically or immunologically equivalent derivative
  • an immunogenic earner protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • modified polypeptides of the invention for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life, resistance to proteolytic degradation in vivo, etc.).
  • modified polypeptides when designed to retain at least one activity of the naturally-occurring form of the protein, are considered "functional equivalents" of the polypeptides described in more detail herein.
  • modified polypeptides may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.
  • polypeptide also provides isolated nucleic acid sequences encoding the polypeptides or polypeptide fragments (referred to in the remainder of this section collectively as "polypeptide"), as well as vectors, host cells, and cultures thereof.
  • polypeptide fragment may be produced using standard polypeptide synthesis methods as will be known to one of skill in the art. Alternatively, such polypeptide fragments, as well as the subject polypeptides, may be produced using recombinant techniques. Chemical synthesis of polypeptides of the invention may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally- assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation.
  • Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-lihked intermediate.
  • the transient thioester-lmked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site.
  • Full length ligation products are chemically identical to proteins produced by cell free synthesis.
  • Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules, (see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr.
  • polypeptide fragments derived from the full-length polypeptides of the invention are isolated peptidyl portions of those polypeptides. Isolated peptidyl portions of those polypeptides may be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such polypeptides. In addition, fragments may be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, proteins may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or may be divided into overlapping fragments of a desired length.
  • the fragments may be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments having a desired property, for example, the capability of functioning as a modulator of the polypeptides of the invention.
  • peptidyl portions of a protein of the invention may be tested for binding activity, as well as inhibitory ability, by expression as, for example, thioredoxin fusion proteins, each of which contains a discrete fragment of a protein of the invention (see, for example, U.S. Patents 5,270,181 and 5,292,646; and PCT publication WO94/ 02502).
  • Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors.
  • suitable vectors for the expression of a polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • the subject nucleic acid is provided in a vector comprising a nucleotide sequence encoding a polypeptide of the invention, and operably linked to at least one regulatory sequence.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
  • the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered.
  • Such vectors may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding a chimeric polypeptide.
  • Approaches include insertion of the nucleic acid in viral vectors including recombinant retroviruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex viruses- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors may be used to transfect cells directly; plasmid DNA may be delivered alone with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers.
  • Nucleic acids may also be directly injected.
  • calcium phosphate precipitation may be carried out to facilitate entry of a nucleic acid into a cell.
  • the subject nucleic acids may be used to cause expression and over-expression of polypeptide of interest in cells propagated in culture, e.g. to produce proteins or polypeptides.
  • This invention also pertains to a host cell transfected with a recombinant gene in order to express a polypeptide of the invention.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a gene comprising a polypeptide of interest may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, insect, plant, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject.
  • Other suitable host cells are known to those skilled in the art.
  • the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide are known to those in the art.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • a polypeptide may be secreted and isolated from a mixture of cells and medium comprising the polypeptide. Alternatively, a polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of a fusion tag.
  • a nucleic acid encoding a polypeptide of the invention is introduced into a host cell, such as by transfection, and the host cell is cultured under conditions allowing expression of the polypeptide.
  • Methods of introducing nucleic acids into prokaryotic and eukaryotic cells are well known in the art. Suitable media for mammalian and prokaryotic host cell culture are well known in the art.
  • the nucleic acid encoding the subject polypeptide is under the control of an inducible promoter, which is induced once the host cells comprising the nucleic acid have divided a certain number of times.
  • IPTG isopropyl beta-D-thiogalactopyranoside
  • a nucleotide sequence encoding all or part of a polypeptide of the invention may be used to produce a recombinant form of a protein via microbial or eukaryotic cellular processes.
  • Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant polypeptides by microbial means or tissue-culture technology in accord with the subject invention.
  • nucleic acid sequences encoding the polypeptides of the invention are further described below.
  • the nucleic acid encoding a polypeptide of the invention is operably linked to a bacterial promoter, e.g., the anaerobic E. coli, NirB promoter or the E. coli lipoprotein Up promoter, described, e.g., in Inouye et al. (1985) Nucl. Acids Res. 13:3101; Salmonella pagC promoter (Miller et al., supra), Shigella ent promoter (Schmitt and Payne, J. Bacteriol.
  • a bacterial promoter e.g., the anaerobic E. coli, NirB promoter or the E. coli lipoprotein Up promoter, described, e.g., in Inouye et al. (1985) Nucl. Acids Res. 13:3101; Salmonella pagC promoter (Miller et al., supra
  • the bacterial promoter can be a constitutive promoter or an inducible promoter.
  • An exemplary inducible promoter is a promoter which is inducible by iron or in iron-limiting conditions. In fact, some bacteria, e.g.,intracellular organisms, are believed to encounter iron-limiting conditions in the host cytoplasm. Examples of iron-regulated promoters of FepA and TonB are known in the art and are described, e.g., in the following references: Headley, N. et al.
  • a signal peptide sequence is added to the construct, such that the polypeptide is secreted from cells.
  • signal peptides are well known in the art.
  • the powerful phage T5 promoter that is recognized by E. coli
  • R ⁇ A polymerase is used together with a lac operator repression module to provide tightly regulated, high level expression or recombinant proteins in E. coli. In this system, protein expression is blocked in the presence of high levels of lac repressor.
  • the D ⁇ A is operably linked to a first promoter and the bacterium further comprises a second D ⁇ A encoding a first polymerase which is capable of mediating transcription from the first promoter, wherein the D ⁇ A encoding the first polymerase is operably linked to a second promoter.
  • the second promoter is a bacterial promoter, such as those delineated above.
  • the polymerase is a bacteriophage polymerase, e.g., SP6, T3, or T7 polymerase and the first promoter is a bacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively.
  • Plasmids comprising bacteriophage promoters and plasmids encoding bacteriophage polymerases can be obtained commercially, e.g., from Promega Corp. (Madison, Wis.) and Nitrogen (San Diego, Calif.), or can be obtained directly from the bacteriophage using standard recombinant D ⁇ A techniques (J. Sambrook, E. Fritsch, T.
  • the bacterium further comprises a D ⁇ A encoding a second polymerase which is capable of mediating transcription from the second promoter, wherein the D ⁇ A encoding the second polymerase is operably linked to a third promoter.
  • the third promoter is a bacterial promoter.
  • more than two different polymerases and promoters could be introduced in a bacterium to obtain high levels of transcription.
  • the use of one or more polymerase for mediating transcription in the bacterium can provide a significant increase in the amount of polypeptide in the bacterium relative to a bacterium in which the DNA is directly under the control of a bacterial promoter.
  • the host cell may include a plasmid which expresses an internal T7 lysozyme, e.g., expressed from plasmid pLysSL (see Examples). Lysis of such host cells liberates the lysozyme which then degrades the bacterial membrane.
  • Other sequences that may be included in a vector for expression in bacterial or other prokaryotic cells include a synthetic ribosomal binding site; strong transcriptional terminators, e.g., to from phage lambda and from the rrnB operon in E. coli, to prevent read through transcription and ensure stability of the expressed polypeptide; an origin of replication, e.g., ColEl; and beta-lactamase gene, conferring ampicillin resistance.
  • Other host cells include prokaryotic host cells. Even more preferred host cells are bacteria, e.g., E. coli. Other bacteria that can be used include Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp., Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp.
  • Hemophilus spp. Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp. Most of these bacteria can be obtained from the American Type Culture Collection (ATCC; 10801 University Boulevard., Manassas, VA 20110- 2209).
  • ATCC American Type Culture Collection
  • YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al, (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83).
  • These vectors may replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
  • drug resistance markers such as ampicillin may be used.
  • mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, P Tk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
  • suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal comprising pBlueBac III).
  • protein production may be achieved using in vitro translation systems.
  • In vitro translation systems are, generally, a translation system which is a cell-free extract comprising at least the minimum elements necessary for translation of an RNA molecule into a protein.
  • An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F).
  • CBP cap-binding protein
  • eIF4F eukaryotic initiation factor 4F
  • in vitro translation systems examples include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, 111.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used.
  • eukaryotic lysates such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla
  • Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any 7 ⁇ ecessary transcription initiation, elongation and termination factors.
  • An RNA nucleotide for in vitro translation may be produced using methods known in the art. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs. When expression of a carboxy terminal fragment of a polypeptide is desired, i.e. a truncation mutant, it may be wecessary to add a start codon (ATG) to the oligonucleotide fragment comprising the desired sequence to be expressed.
  • ATG start codon
  • MAP methionine aminopeptidase
  • removal of an N-terminal methionine may be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al.).
  • a host which produces MAP e.g., E. coli or CM89 or S. cerevisiae
  • purified MAP e.g., procedure of Miller et al.
  • the expression of a polypeptide may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature, 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, ⁇ MBO J., 3:1671-1680; Broglie et al., 1984, Science, 224:838-843); or heat shock promoters, eg., soybean hsp 17.5- ⁇ or hsp 17.3-B (Gurley et al., 1986, Mol.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the PGHS-2 sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • the DNA encoding the subject polypeptide is cloned into the pBlueBacIII recombinant transfer vector (Invitrogen, San Diego, Calif.) downstream of the polyhedrin promoter and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.) to generate recombinant virus.
  • pBlueBacIII recombinant transfer vector Invitrogen, San Diego, Calif.
  • Sf9 insect cells derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.
  • the subject polypeptides are prepared in transgenic animals, such that in certain embodiments, the polypeptide is secreted, e.g., in the milk of a female animal.
  • Viral vectors may also be used for efficient in vitro introduction of a nucleic acid into a cell. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, polypeptides encoded by genetic material in the viral vector, e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into mammals. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • a major prerequisite for the use of retro viruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the antisense E6AP constructs, rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.
  • retroviruses include pLJ, pZJ-P, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include Crip, Cre, 2 and Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al.
  • retroviral vectors as a gene delivery system for nucleic acids encoding the subject polypeptides, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the genetic material, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be beneficial in circumstances wherein the tissue (e.g., nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.
  • tissue e.g., nontransformed cells
  • retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle.
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079- 9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al.
  • Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single-chain antibody/env chimeric proteins).
  • a protein or other variety e.g., lactose to convert the env protein to an asialoglycoprotein
  • chimeric proteins e.g., single-chain antibody/env chimeric proteins.
  • retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the genetic material of the retroviral vector.
  • adenovirus-derived vectors The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non- dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).
  • the virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj- Ahmand and Graham (1986) J. Virol. 57:267).
  • adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127).
  • Expression of the inserted genetic material can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
  • MLP major late promoter
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • Adeno-associated virus is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al.,(1992) Am. J. Respir. Cell. Mol. Biol.
  • Vectors comprising as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251- 3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.
  • viral vector systems may be derived from herpes virus, vaccinia virus, and several RNA viruses.
  • non-viral methods can also be employed to cause expression of nucleic acids encoding the subject polypeptides, e.g. in a cell in vitro or in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of genetic material by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.
  • genetic material can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and, optionally, which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • lipofection of papilloma-infected cells can be carried out using liposomes tagged with monoclonal antibodies against PV-associated antigen (see Viac et al. (1978) J Invest Dermatol 70:263- 266; see also Mizuno et al. (1992) Neurol. Med. Chir. 32:873-876).
  • the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180).
  • a gene binding agent such as polylysine
  • genetic material encoding the subject chimeric polypeptides can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S. Patent 5,166,320).
  • adenovirus or fiisogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-comprising endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano et al. (1993) PNAS 90:2122).
  • D. Antigens The polypeptides of the invention, or fragments thereof may comprise M. tuberculosis antigens. Antigens generally have the ability to induce an immunogenic response.
  • antigens have the ability to induce proliferation and/or cytokine production (i.e., interferon-.gamma. and/or interleukin-12 production) in T cells, NK cells, B cells and/or macrophages derived from an M. tuberculosis-immune individual.
  • cytokine production i.e., interferon-.gamma. and/or interleukin-12 production
  • the selection of cell type for use in evaluating an immunogenic response to a antigen will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing B cells and/or macrophages.
  • An M. tuberculosis- immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M.
  • tuberculosis i.e., substantially free of disease symptoms.
  • Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD) and an absence of any signs or symptoms of tuberculosis disease.
  • PPD tuberculosis proteins
  • T cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods known to those of ordinary skill in the art. For example, a preparation of PBMCs (i.e., peripheral blood mononuclear cells) may be employed without further separation of component cells.
  • PBMCs may generally be prepared, for example, using density centrifugation through "FICOLL” (Winthrop Laboratories, N.Y.).
  • T cells for use in the assays described herein may also be purified directly from PBMCs.
  • an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins may be employed.
  • Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosis-immune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells.
  • T cells may then be cloned and tested with individual proteins, using methods known to those of ordinary skill in the art, to more accurately define individual T cell specificity.
  • antigens that test positive in assays for proliferation and/or cytokine production i.e., interferon-.gamma. and/or interleukin-12 production
  • T cells NK cells
  • B cells i.e., interferon-.gamma. and/or interleukin-12 production
  • macrophages derived from an M. tuberculosis-immune individual are considered immunogenic.
  • Such assays may be performed, for example, using the representative procedures described below. Immunogenic portions of such antigens, e.g. "antigenically active fragments,” may be identified using similar assays, and may be present within the polypeptides described herein.
  • the ability of a polypeptide (e.g., an immunogenic antigen, or a portion or other variant thereof) to induce cell proliferation may be evaluated, for example, by contacting the cells (e.g., T cells and/or NK cells) with the polypeptide and measuring the proliferation of the cells.
  • the amount of polypeptide that is sufficient for evaluation of about 10. sup.5 cells ranges from about 10 ng/mL to about 100 .mu.g/mL and preferably is about 10 .mu.g/mL.
  • the incubation of polypeptide with cells is typically performed at 37. degree. C. for about six days.
  • the cells are assayed for a proliferative response, which may be evaluated by methods known to those of ordinary skill in the art, such as exposing cells to a pulse of radiolabeled thymidine and measuring the incorporation of label into cellular DNA.
  • a polypeptide that results in at least a three fold increase in proliferation above background i.e., the proliferation observed for cells cultured without polypeptide
  • the ability of a polypeptide to stimulate the production of interferon-.gamma. and/or interleukin-12 in cells may be evaluated, for example, by contacting the cells with the polypeptide and measuring the level of interferon-.gamma.
  • the amount of polypeptide that is sufficient for the evaluation of about 10.sup.5 cells ranges from about 10 ng/mL to about 100 .mu.g/mL and preferably is about 10 .mu.g/mL.
  • the polypeptide may be, but need not be, immobilized on a solid support, such as a bead or a biodegradable microsphere, such as those described in U.S. Pat. Nos. 4,897,268 and 5,075,109.
  • the incubation of polypeptide with the cells is typically performed at 37.degree. C. for about six days. Following incubation with polypeptide, the cells are assayed for interferon-.gamma.
  • interleukin-12 and/or interleukin-12 (or one or more subunits thereof), which may be evaluated by methods known to those of ordinary skill in the art, such as an enzyme-linked immunosorbent assay (ELISA) or, in the case of IL-12 P70 subunit, a bioassay such as an assay measuring proliferation of T cells, hi general, a polypeptide that results in the production of at least 50 pg of interferon-.gamma. per mL of cultured supernatant (containing 10.sup.4 -10. sup.5 T cells per mL) is considered able to stimulate the production of interferon-.gamma..
  • ELISA enzyme-linked immunosorbent assay
  • a bioassay such as an assay measuring proliferation of T cells, hi general, a polypeptide that results in the production of at least 50 pg of interferon-.gamma. per mL of cultured supernatant (containing 10.sup.4 -10. sup.
  • a polypeptide that stimulates the production of at least 10 pg mL of IL-12 P70 subunit, and/or at least 100 pg/mL of IL-12 P40 subunit, per 10.sup.5 macrophages or B cells (or per 3. times.10. sup.5 PBMC) is considered able to stimulate the production of IL-12.
  • immunogenic antigens are those antigens that stimulate proliferation and/or cytokine production (i.e., interferon-.gamma. and/or interleukin-12 production) in T cells, NK cells, B cells and/or macrophages derived from at least about 25% of M. tuberculosis-immune individuals.
  • cytokine production i.e., interferon-.gamma. and/or interleukin-12 production
  • polypeptides having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed.
  • antigens having superior therapeutic properties will not stimulate proliferation and/or cytokine production in vitro in cells derived from more than about 25%, of individuals who are not M.
  • tuberculosis-immune thereby eliminating responses that are not specifically due to M. tuberculosis-responsive cells.
  • Those antigens that induce a response in a high percentage of T cell, NK cell, B cell and/or macrophage preparations from M. tuberculosis-immune individuals have superior therapeutic properties.
  • Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use on experimental animals are described in detail below. Efficacy may be determined based on the ability of the antigen to provide at least about a 50% reduction in bacterial numbers and/or at least about a 40% decrease in mortality following experimental infection. Suitable experimental animals include mice, guinea pigs and primates.
  • Immunogenic Compositions and Vaccines and Methods of Use 1. Immunogenic Compositions and Vaccines Comprising M. tuberculosis Strains or Recombinant Antigens
  • the necrosis-deficient tuberculosis strains and/or the polypeptides of the invention may comprise immunogenic compositions and vaccines.
  • a strain of the invention may be attenuated in virulence and/or retains its immunogenic properties without being able to cause a tuberculosis infection.
  • a polypeptide antigen of the present invention may correspond to a protein that is essential for virulence or infectivity of tuberculosis bacteria.
  • such strains and polypeptides may be used as part of an immunogenic composition or vaccine, for example, formulated in a pharmaceutically acceptable carrier, to prevent tuberculosis infection.
  • Such immunogenic compositions or vaccines may further comprise other known tuberculosis vaccines, such as, for example, Bacillus Calmette-Guerin, or combinations of the strains and polypeptides of the present invention. In certain embodiments such compositions are used for immunization against M. tuberculosis.
  • a vaccine comprises an immunoprotective and non-toxic amount of a mutant strain of the invention. Suitable amounts can be determined by the person skilled in the art and are typically 10.sup.7 to 10.sup.9 bacteria.
  • a polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue.
  • a vaccine comprises an immunoprotective and non-toxic amount of an antigen of the invention. Purified or partially purified antigenic polypeptides or fragments thereof may be formulated as a vaccine or immunogenic composition.
  • the amount of polypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 .mu.g. Suitable dose range will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • an effective dose of the strain or polypeptide for inducing an immune response in a subject is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • An effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve an induction of an immune response using techniques that are well known in the art.
  • Dosage amount and interval may be adjusted individually.
  • the polypeptides and/or strains of the invention may be administered in about 1 to 3 doses for a 1-36 week period.
  • a suitable dose is an amount of polypeptide or strain that, when administered as described above, is capable of raising an immune response in an immunized patient sufficient to protect the patient from tuberculosis infection for at least 1- 2 years.
  • compositions may also include adjuvants to enhance immune responses.
  • proteins may be further suspended in an oil emulsion to cause a slower release of the proteins in vivo upon injection.
  • the optimal ratios of each component in the formulation may be determined by techniques well known to those skilled in the art.
  • adjuvants may be employed in the vaccines of this invention to enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a specific or nonspecific stimulator of immune responses, such as lipid A, or Bortadella pertussis.
  • Suitable adjuvants are commercially available and include, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).
  • Suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A, quil A, SBASlc, SBAS2 (Ling et al., 1997, Vaccine 15:1562-1567), SBAS7, Al(OH) 3 and CpG oligonucleotide (WO96/02555).
  • the adjuvant may induce an immune response comprising Thl aspects.
  • Suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminum salt.
  • An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of 3D-MLP and the saponin QS21 as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.
  • Nucleic acids of the invention encoding immunogenic polypeptides and fragments thereof, may comprise also immunogenic compositions and vaccines.
  • the subject nucleic acds may be used to form nucleic acid vaccines, e.g., DNA vaccines, for immunization against tuberculosis.
  • DNA vaccination presents a number of features of potential value. Multiple antigens may included simultaneously in the vaccination. Such vaccination may work even in the presence of maternal antibodies.
  • nucleic acid of the invention in genetic immunization may employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein carriers (Wu et al., J Biol Chem.
  • DNA vaccination may be applied to eliminate or ameliorate existing disease or conditions, including chronic infectious diseases.
  • the subject DNA vaccines may be used for immunizing subjects against such infections as HSV, HIV, HCV, influenza, malaria, Ebola, hepatitis B, pappillomavirus and the like.
  • the DNA vaccines may also be employed as part of a protocol for induction of tolerance, such as in the treatment of allergies and other autoimmune conditions, such as multiple sclerosis, Type I diabetes, and rheumatoid arthritis.
  • the goal of vaccination is the induction of protective immunity.
  • the target was once limited to infectious diseases, but has now broadened to include treatment of tumors, allergy, and even autoimmune diseases.
  • the delivery of naked plasmid DNA results in the expression of the encoded antigen by muscle cells, and perhaps APCs, resulting in the induction of protective CTLs as well as antibody responses.
  • This method of "genetic immunization" with polynucleic acid vaccines (PNV) may represent a significant advance in vaccination technology because it may be used repeatedly to immunize to different antigens while avoiding the risk of an infectious virus and the problem of the immune response to the vector.
  • DNA vaccination using the nucleic acids of the present invention may produce different results from other vaccination efforts using DNA, such as naked injection of DNA.
  • the pattern of antigen express, both temporally and spatially, may differ from naked injection of DNA.
  • the nucleic acids of the present invention may be used to deliver a coding sequence for an antigen(s) as part of a genetic immunization protocol.
  • U.S. Patent No. 5,783,567 and WO 94/04171 present a number of potential polypeptide sequences for inducing an immunogenic response.
  • the subject nucleic acids may elicit a strong immune response even at low dose.
  • the choice of components with which the nucleic acids are formulated, along with selection of regulatory elements, may be used to optimize the vaccine response.
  • the material in which the nucleic acid or other material is incorporated may serve as an adjuvant. Additional adjuvants may be administered, for example, within the composition or in conjunction with the composition to enhance the inherent adjuvant effect of the compositions By controlling the rate of release of the sequence giving rise to the antigen, it may be possible to prepare a single dose vaccine to replace a vaccination protocol requiring an initial vaccination followed by booster doses.
  • a variety of DNA vaccination techniques may be employed to elicit a stronger immune response.
  • a naked nucleic acid such as DNA
  • a composition of the present invention loaded with the same nucleic acid or, alternatively, a different nucleic acid or acids (as well as possibly other materials).
  • the initial dose of naked nucleic acid followed by release of nucleic acid from the composition may result in a more effective vaccination.
  • the subject method may be used as part of a vaccination against microbial pathogens.
  • CTLs cytotoxic T-lymphocytes
  • CTLs kill virally- or bacterially-infected cells when their T cell receptors recognize foreign peptides associated with MHC class I and/or class II molecules. These peptides may be derived from endogenously synthesized foreign proteins, regardless of the protein's location or function within the pathogen. By recognition of epitopes from conserved proteins, CTLs may provide heterologous protection. In the case of intracellular bacteria, proteins secreted by or released from the bacteria are processed and presented by MHC class I and II molecules, thereby generating T-cell responses that may play a role in reducing or eliminating infection.
  • the subject method may be used to produce a protective vaccination against infection by Mycobacterium tuberculosis.
  • Genes encoding Mycobacterium tuberculosis proteins such as, for example, Rv3351c, Rv3875 and Rv3874, may cloned into eukaryotic expression vectors, and formulated for expression of the encoded proteins in mammalian muscle cells in vivo.
  • DNA vaccination may use mucosal delivery, which allows for easy administration, reduced side-effects, and the possibility of frequent boosting without requiring trained medical personnel.
  • Mucosal delivery of vaccines appears to be the only effective means of inducing immune responses in the mucosal secretions.
  • many pathogens enter the body through the mucosal tissues of the gut or the respiratory or genital tracts 3.
  • Formulations containing a strain or antigen or nucleic acid of the present invention may be administered to a subject per se or in the form of a pharmaceutical or therapeutic composition.
  • Pharmaceutical compositions comprising the proteins may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the polypeptides into preparations which can be used pharmaceutically.
  • Pharmaceutically acceptable carriers, suitable neutralizing buffers, and suitable delivering systems can be selected by the person skilled in the art. Proper formulation is dependent upon the route of administration chosen.
  • the mode of administration of the vaccines of the present invention may be any suitable route which delivers an immunoprotective amount of the vaccine to the subject.
  • the vaccine is most commonly administered orally or intranasally.
  • the proteins may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • the proteins may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • a composition can be readily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art. Such carriers enable the proteins to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
  • the proteins may take the form of tablets, lozenges, etc. formulated in conventional manner.
  • the strains or polypeptides for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the proteins and a suitable powder base such as lactose or starch.
  • the strains or polypeptides may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the strains or polypeptides may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the proteins may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver an antigen.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the strains or polypeptides may also be encapsulated in microspheres (U.S. Pat. Nos. 5,407,609; 5,853,763; 5,814,344 and 5,820,883).
  • they may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic or vaccinating agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the material for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the reagent, additional strategies for stabilization may be employed.
  • compositions and vaccines of the present invention may be used in methods of treating or preventing tuberculosis in mammals, for example, humans.
  • a method for eliciting an immunogenic response in a mammal may comprise administering to a mammal an effective amount of a M. tuberculosis strain of the invention.
  • a method for eliciting an immunogenic response in a mammal may comprise administering to an subject at least one polypeptide or antigenically active fragment thereof having for example, at least about 80%,, at least about 85%,, at least about 90%,, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding polypeptide or fragment thereof encoded by a gene from M. tuberculosis involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • such gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • a method for eliciting an immunogenic response in a mammal may comprise administering to an subject a nucleic acid encoding at least one antigenically active gene product, for example a polypeptide or antigenically active fragment thereof, having for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding gene product encoded by a gene from M.
  • the nucleic acid sequence may be comprised of DNA or RNA, and may optionally comprise a vector.
  • the gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • such methods may comprise a method of treating tuberculosis.
  • the above-described methods may further serve to vaccinate said mammal rather than simply elicit an immunogenic response.
  • a method for vaccinating a mammal against tuberculosis may comprise in certain embodiments administering to an subject at least one polypeptide or antigenically active fragment thereof having for example, at least about 80%>, at least about 85%>, at least about 90%>, at least about 95%o, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding polypeptide or fragment thereof encoded by a gene from, for example, M. tuberculosis involved in tuberculosis-induced epithelial cell permeation and/or necrosis.
  • a method for vaccinating a mammal against tuberculosis comprises administering to an subject a nucleic acid encoding at least one antigenically active gene product, for example a polypeptide or antigenically active fragment thereof, having for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 91%, at least about 98%,, or at least about 99%, identity to the corresponding gene product encoded by a gene from, for example, M.
  • at least one antigenically active gene product for example a polypeptide or antigenically active fragment thereof, having for example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 91%, at least about 98%,, or at least about 99%, identity to the corresponding gene product encoded by a gene from, for example, M.
  • the nucleic acid sequence may be comprised of DNA or RNA, and may optionally comprise a vector.
  • the gene may be selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877.
  • Such methods may elicit an immunogenic response that leads to protective immunity.
  • such methods may comprise a method of treating tuberculosis.
  • the polypeptides and fragments of the invention are antigens. Still further, the present invention provides antibodies specific to such antigens. In certain embodiments, the antibodies are specific for an antigenic polypeptide or antigenically active fragment thereof encoded by a gene selected from the group consisting of: Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877. In certain embodiments, an antibody may be specific for a Rv3351c polypeptide or fragment thereof. In other embodiments, an antibody may be specific for the polypeptide encoded by SEQ ID NO: 1, or an antigenically active fragment of said polypeptide.
  • peptides based on a polypeptide of the invention e.g., having an amino acid sequence of SEQ ID NO: 1 or an immunogenic fragment thereof
  • antisera or monoclonal antibodies may be made using standard methods.
  • An exemplary immunogenic fragment may contain eight, ten or more consecutive amino acid residues of SEQ ID NO: 1.
  • the present invention contemplates a purified antibody that binds specifically to a polypeptide of the invention and which does not substantially cross-react with a protein which is less than about 80%, or less than about 90%, identical to SEQ ID NO: 1.
  • the present invention contemplates an array comprising a substrate having a plurality of address, wherein at least one of the addresses has disposed thereon a purified antibody that binds specifically to a polypeptide of the invention.
  • Antibodies may be elicited by methods known in the art.
  • a mammal such as a mouse, a hamster or rabbit may be immunized with an immunogenic form of a polypeptide of the invention (e.g., an antigenic fragment which is capable of eliciting an antibody response).
  • immunization may occur by using a nucleic acid of the acid, which presumably in vivo expresses the polypeptide of the invention giving rise to the immunogenic response observed.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • a peptidyl portion of a polypeptide of the invention may be administered in the presence of adjuvant.
  • the progress of immunization may be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays may be used with the immunogen as antigen to assess the levels of antibodies.
  • antibody producing cells may be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the polypeptides of the invention and the monoclonal antibodies isolated.
  • Antibodies directed against the polypeptides of the invention can be used to selectively block the action of the polypeptides of the invention.
  • Antibodies against a polypeptide of the invention may be employed to treat infections, particularly bacterial infections and diseases.
  • the present invention contemplates a method for treating a subject suffering from a M. tuberculosis related disease or disorder, comprising administering to an animal having the condition a therapeutically effective amount of a purified antibody that binds specifically to a polypeptide of the invention, i another example, the present invention contemplates a method for inhibiting SEQ ID NO: 1 dependent growth or infectivity of M. tuberculosis, comprising contacting M.
  • tuberculosis with a purified antibody that binds specifically to a polypeptide of the invention.
  • antibodies reactive with a polypeptide of the invention are used in the immunological screening of cDNA libraries constructed in expression vectors, such as ⁇ gtll, ⁇ gtl8-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, ⁇ gtl 1 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of a polypeptide of the invention can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from phage infected bacterial plates with an antibody specific for a polypeptide of the invention. Phage scored by this assay can then be isolated from the infected plate. Thus, homologs of a polypeptide of the invention can be detected and cloned from other sources.
  • Antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
  • the antibodies of the invention, or variants thereof are modified to make them less immunogenic when administered to a subject.
  • the antibody may be "humanized"; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al. (1991) Biotechnology 9, 266-273.
  • transgenic mice, or other mammals may be used to express humanized antibodies. Such humanization may be partial or complete.
  • Diagnosis of tuberculosis is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative).
  • Tuberculin PPD protein-purified derivative
  • Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
  • a method may comprise: detecting in a sample the presence of at least one polypeptide encoded by a M. tuberculosis gene selected from the group consisting of:Rv3351c, Rv3875, Rv3874, Rv3871, Rv3876 and Rv3877. Detection may be accomplished, for example, by an antibody specific for the polypeptide to be detected.
  • the invention further provides a method for detecting the presence of M. tuberculosis in a biological sample. Detection of M. tuberculosis in a subject, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a M. tuberculosis related disease or disorder.
  • the method involves contacting the biological sample with a compound or an agent capable of detecting a polypeptide of the invention or a nucleic acid of the invention.
  • biological sample when used in reference to a diagnostic assay is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the detection method of the invention may be used to detect the presence of M. tuberculosis in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of a nucleic acid of the invention include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of polypeptides of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunofluorescence, radioimmunoassays and competitive binding assays.
  • ELISAs enzyme linked immunosorbent assays
  • polypeptides of the invention can be detected in vivo in a subject by introducing into the subject a labeled antibody specific for a polypeptide of the invention.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. It may be possible to use all of the diagnostic methods disclosed herein for pathogens in addition to M. tuberculosis.
  • the present invention contemplates a method for detecting the presence of tuberculosis bacteria, for example, M.
  • tuberculosis in a sample, the method comprising: (a) providing a sample to be tested for the presence of tuberculosis bacteria; (b) contacting the sample with an antibody that binds specifically to a polypeptide of the invention or fragment thereof under conditions which permit association between the antibody and its ligand; and (c) detecting interaction of the antibody with its ligand, thereby detecting the presence of tuberculosis bacteria in the sample.
  • the present invention contemplates a method for detecting the presence of tuberculosis bacteria, for example, M. tuberculosis, in a sample, the method comprising: (a) providing a sample to be tested for the presence of tuberculosis bacteria; (b) contacting the sample with an antibody reactive against at least about eight consecutive amino acid residues of a polypeptide encoded by SEQ ID NO: 1 under conditions which permit association between the antibody and its ligand; and (c) detecting interaction of the antibody with its ligand, thereby detecting the presence of tuberculosis bacteria in the sample.
  • tuberculosis bacteria for example, M. tuberculosis
  • the present invention contemplates a method for diagnosing a patient suffering from tuberculosis, comprising: (a) obtaining a biological sample from a patient; (b) detecting the presence or absence of a polypeptide of the invention, or a nucleic acid encoding a polypeptide of the invention, in the sample; and (c) diagnosing a patient suffering from a M. tuberculosis related disease or disorder based on the presence of a polypeptide of the invention, or a nucleic acid encoding a polypeptide of the invention, in the patient sample.
  • this invention provides methods for using one or more of the polypeptides to diagnose tuberculosis using a skin test in vivo.
  • a skin test is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above.
  • DTH delayed-type hypersensitivity
  • Such injection may be achieved using any suitable device sufficient to contact the polypeptide with dermal cells of the patient, such as, for example, a tuberculin syringe or 1 mL syringe.
  • the reaction is measured at least about 48 hours after injection, more preferably about 48 to about 72 hours after injection.
  • the DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen (i.e., the immunogenic portion of the polypeptide employed, or a variant thereof).
  • the response may be measured visually, using a ruler.
  • a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter is a positive response, indicative of tuberculosis infection, which may or may not be manifested as an active disease.
  • polypeptides of this invention are preferably formulated, for use in a skin test, as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier.
  • Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 .mu.g to about 100 .mu.g, preferably from about 10 .mu.g to about 50 .mu.g in a volume of 0.1 mL.
  • the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80.TM.
  • the present invention provides methods for using the polypeptides to diagnose tuberculosis, hi this aspect, methods are provided for detecting M. tuberculosis infection in a biological sample using the polypeptides alone or in combination.
  • a biological sample is any antibody-containing sample obtained from a patient.
  • the sample is whole blood, sputum, serum, plasma, saliva cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply.
  • the polypeptide(s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide(s) in the sample relative to a predetermined cut-off value. The presence of such antibodies indicates previous sensitization to mycobacterial antigens which may be indicative of tuberculosis.
  • the polypeptides used are preferably complementary (i.e., one component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide).
  • Complementary polypeptides may generally be identified by using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with M. tuberculosis. After determining which samples test positive (as described below) with each polypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. Such polypeptides are complementary. Approximately 25-30% of sera from tuberculosis- infected individuals are negative for antibodies to any single protein.
  • Complementary polypeptides may, therefore, be used in combination to improve sensitivity of a diagnostic test.
  • assay formats known to those of ordinary skill in the art for using one or more polypeptides to detect antibodies in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference.
  • Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Nucleic acids, e.g., DNA and RNA, may be used directly for detection or may be amplified, e.g., enzymatically by using PCR or other amplification technique, prior to analysis. Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence.
  • Point mutations can be identified by hybridizing a nucleic acid, e.g., amplified DNA, to a nucleic acid of the invention, which nucleic acid may be labeled. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g. Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
  • Agents for detecting a nucleic acid of the invention include labeled or labelable nucleic acid probes capable of hybridizing to a nucleic acid of the invention.
  • the nucleic acid probe can comprise, for example, the full length sequence of a nucleic acid of the invention, or an equivalent thereof, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions.
  • Agents for detecting a polypeptide of the invention include labeled or labelable antibodies capable of binding to a polypeptide of the invention.
  • Antibodies may be polyclonal, or alternatively, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • Labeling the probe or antibody also encompasses direct labeling of the probe or antibody by coupling (e.g., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • detection of a nucleic acid of the invention in a biological sample involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which can be particularly useful for distinguishing between orthologs of polynucleotides of the invention (see Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a nucleic acid of the invention under conditions such that hybridization and amplification of the polynucleotide (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • the diagnostic assays of the invention may also be used to monitor the effectiveness of an anti- tuberculosis treatment in an individual suffering from an M. tuberculosis related disease or disorder.
  • the presence and/or amount of a nucleic acid of the invention or a polypeptide of the invention can be detected in an individual suffering from a M. tuberculosis related disease or disorder before and after treatment with anti-M. tuberculosis therapeutic agent.
  • Any change in the level of a polynucleotide or polypeptide of the invention after treatment of the individual with the therapeutic agent can provide information about the effectiveness of the treatment course.
  • no change, or a decrease, in the level of a polynucleotide or polypeptide of the invention present in the biological sample will indicate that the therapeutic is successfully combating the M. tuberculosis related disease or disorder.
  • kits for detecting the presence of M. tuberculosisin a biological sample can comprise a labeled or labelable compound or agent capable of detecting a polynucleotide or polypeptide of the invention in a biological sample; means for determining the amount of M. tuberculosisin the sample; and means for comparing the amount of M. tuberci ⁇ osis in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect a polynucleotide or polypeptide of the invention.
  • Modulators to polypeptides of the invention and other structurally related molecules, and complexes containing the same, may be identified and developed as set forth below and otherwise using techniques and methods known to those of skill in the art.
  • the modulators of the invention may be employed, for instance, to inhibit and treat tuberculosis.
  • exemplary methods involve contacting M. tuberculosis with a polypeptide of the invention that modulates the same or another polypeptide from such pathogen, a nucleic acid encoding such polypeptide of the invention, or a compound thought or shown to be effective against such pathogen.
  • the present invention contemplates a method for treating a patient suffering from an infection of M. tuberculosis, comprising administering to the patient an amount of a Rv3351c inhibitor effective to inhibit the expression and/or activity of a polypeptide of the invention.
  • the animal is a human or a livestock animal such as a cow, pig, goat or sheep.
  • the present invention further contemplates a method for treating a subject suffering from a M. tuberculosis related disease or disorder, comprising administering to an animal having the condition a therapeutically effective amount of a molecule identified using one of the methods of the present invention.
  • the present invention contemplates making any molecule that is shown to modulate the activity of any polypeptide of the invention.
  • compounds may be selected from the following classes of compounds: proteins, peptides, peptidomimetics, or small molecules.
  • the compounds may be selected from the following classes of compounds: antisense nucleic acids, small molecules, polypeptides, proteins including antibodies, peptidomimetics, or nucleic acid analogs.
  • the compounds may be selected from a library of compounds. These libraries may be generated using combinatorial synthetic methods.
  • the ability of a compound to bind or modulate the activity of a gene or protein may be determined by using any of a variety of suitable assays.
  • the ability of a compound to bind or modulate a target protein or gene may be evaluated by an in vitro assay.
  • the assay may be an in vivo assay.
  • Such assays are well-known to one of skill in the art and, based on the present description, may be adapted to the methods of the present invention with no more than routine experimentation. All of the screening methods described in this section may be accomplished by using a variety of assay formats.
  • the assays may identify agents, e.g., drugs, which are either agonists or antagonists of expression of a target gene of interest, or of a protei protein or protein-substrate interaction of a target of interest, or of the role of target gene products in the pathogenesis of normal or abnormal cellular physiology, proliferation, and/or differentiation and disorders related thereto.
  • agents e.g., drugs, which are either agonists or antagonists of expression of a target gene of interest, or of a protei protein or protein-substrate interaction of a target of interest, or of the role of target gene products in the pathogenesis of normal or abnormal cellular physiology, proliferation, and/or differentiation and disorders related thereto.
  • Assay formats which approximate such conditions as formation of protein complexes or protein-nucleic acid complexes, enzymatic activity, and even specific signaling pathways, may be generated in many different forms, as those skilled in the art will appreciate based on the present description and include but are not limited to assays based on cell-free systems, e.g., purified proteins or cell lysates, as well as cell-based assays which utilize intact cells.
  • Assays of the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often used as primary screens as they may be generated to permit rapid development and often easy detection of an alteration in a molecular target which is mediated by a test compound.
  • the effects of cellular toxicity and/or bioavailability of the test compound may be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target.
  • potential modifiers e.g., activators or inhibitors of protein-substrate, protein-protein interactions or nucleic acid- protein interactions of interest may be detected in a cell-free assay generated by constitution of function interactions of interest in a cell lysate.
  • the assay may be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.
  • binding assays may be used to detect agents which, by binding to an active site of a protein, or by disrupting the binding of protein-protein interactions or protein-nucleic acid interactions or the subsequent binding of such a complex or individual protein or nucleic acid to a substrate, may inhibit signaling or other effects resulting from the given interaction.
  • drugs may be developed which modulate the activity of the first polypeptide by modulating its binding to the second polypeptide (referred to herein as a "binding partner” or “binding partner”).
  • Cell-free assays may be used to identify compounds which are capable of interacting with a polypeptide or binding partner, to thereby modify the activity of the polypeptide or binding partner. Such a compound may, e.g., modify the structure of the polypeptide or binding partner and thereby effect its activity.
  • Cell-free assays may also be used to identify compounds which bind a polypeptide or modulate the interaction between a polypeptide and a binding partner.
  • cell-free assays for identifying such compounds consist essentially in a reaction mixture containing a polypeptide and a test compound or a library of test compounds in the presence or absence of a binding partner.
  • a test compound may be, e.g., a derivative of a binding partner, e.g., a biologically inactive peptide, or a small molecule.
  • Agents to be tested for their ability to act as interaction inhibitors may be produced, for example, by bacteria, yeast or other organisms (e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly.
  • the candidate therapeutic agent is a small organic molecule.
  • the present invention provides assays that may be used to screen for agents which modulate protein-protein interactions, nucleic acid-protein interactions, or protein-substrate interactions.
  • the screening assays of the present invention may be designed to detect agents which disrupt binding of protein-protein interaction binding moieties.
  • the subject assays will identify inhibitors of the enzymatic activity of a protein or protein-protein interaction complex.
  • the compound is a mechanism based inhibitor which chemically alters one member of a protein-protein interaction or one chemical group of a protein and which is a specific inhibitor of that member, e.g., has an inhibition constant 10-fold, 100-fold, or 1000-fold different compared to homologous proteins.
  • assays which detect inhibitory agents on the basis of their ability to interfere with binding of components of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.
  • the compound of interest is contacted with a mixture generated from protein-protein interaction component polypeptides. Detection and quantification of expected activity from a given protein-protein interaction provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the two polypeptides.
  • the efficacy of the compound may be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay may also be performed to provide a baseline for comparison. In the control assay, the formation of complexes is quantitated in the absence of the test compound.
  • Complex formation between component polypeptides, polypeptides and genes, or between a component polypeptide and a substrate may be detected by a variety of techniques, many of which are effectively described above. For instance, modulation in the formation of complexes may be quantitated using, for example, detectably labeled proteins (e.g., radiolabeled, fluorescently labeled, or enzymatically labeled), by immunoassay, or by chromatographic detection.
  • detectably labeled proteins e.g., radiolabeled, fluorescently labeled, or enzymatically labeled
  • immunoassay e.g., immunoassay
  • chromatographic detection e.g., chromatographic detection.
  • One aspect of the present invention provides reconstituted protein preparations, e.g., combinations of proteins participating in protein-protein interactions. Such methods are referred to within this section as in vitro.
  • one exemplary screening assay of the present invention includes the steps of contacting a polypeptide or functional fragment thereof or a binding partner with a test compound or library of test compounds and detecting the formation of complexes.
  • the molecule may be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker.
  • Interaction of a test compound with a polypeptide or fragment thereof or binding partner may then be detected by determining the level of the two labels after an incubation step and a washing step. The presence of two labels after the washing step is indicative of an interaction.
  • An interaction between molecules may also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants.
  • a library of test compounds may be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the polypeptide, functional fragment thereof, polypeptide analog or binding partner is then flown continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.
  • Another exemplary assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) a polypeptide, (ii) a binding partner, and (iii) a test compound; and (b) detecting interaction of the polypeptide and the binding partner.
  • the polypeptide and binding partner may be produced recombinantly, purified from a source, e.g., plasma, or chemically synthesized, as described herein.
  • the compounds of this assay may be contacted simultaneously.
  • a polypeptide may first be contacted with a test compound for an suitable amount of time, following which the binding partner is added to the reaction mixture.
  • the efficacy of the compound may be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay may also be performed to provide a baseline for comparison. In the control assay, isolated and purified polypeptide or binding partner is added to a composition containing the binding partner or polypeptide, and the formation of a complex is quantitated in the absence of the test compound.
  • Complex formation between a polypeptide and a binding partner may be detected by a variety of techniques. Modulation of the formation of complexes may be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners
  • immunoassay or by chromatographic detection.
  • polypeptide or its binding partner may be immobilize to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of polypeptide to a binding partner, may be accomplished in any vessel suitable for containing the reactants. Examples include microtifre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein may be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the binding partner e.g., an 35s-labeled binding partner
  • the test compound glutathione derivatized microtitre plates
  • the binding partner e.g., an 35s-labeled binding partner
  • the test compound glutathione derivatized microtitre plates
  • the binding partner e.g., an 35s-labeled binding partner
  • the test compound glutathione derivatized microtitre plates
  • the mixture incubated under conditions conducive to complex formation, e.g., at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g., beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated.
  • the complexes may be dissociated from the matrix, separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and the level of polypeptide or binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • polypeptide or its cognate binding partner may be immobilized utilizing conjugation of biotin and streptavidin.
  • biotinylated polypeptide molecules may be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with the polypeptide may be derivatized to the wells of the plate, and polypeptide trapped in the wells by antibody conjugation.
  • preparations of a binding partner and a test compound are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well may be quantitated.
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with polypeptide and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity.
  • the enzyme may be chemically conjugated or provided as a fusion protein with the binding partner.
  • the binding partner may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex may be assessed with a chromogenic substrate of the enzyme, e.g., 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l- napthol.
  • a fusion protein comprising the polypeptide and glutathione-S- transferase may be provided, and complex formation quantitated by detecting the GST activity using 1 -chloro-2,4-dinitrobenzene.
  • antibodies against the protein such as anti-polypeptide antibodies
  • the protein to be detected in the complex may be "epitope tagged" in the form of a fusion protein which includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g., from commercial sources).
  • the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety.
  • myc-epitopes which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc., New Haven, CT) or the pEZZ-protein A system (Pharmacia, NJ).
  • the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins.
  • semi- purified it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins.
  • the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and may be present at 90-95%> purity.
  • the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein-protein interaction, or nucleic acid-protein interaction.
  • the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions.
  • the system may be derived to favor discovery of inhibitors of particular intermediate states of the protein-protein interaction.
  • a reconstituted protein assay may be carried out both in the presence and absence of a candidate agent, thereby allowing detection of an inhibitor of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.
  • Assaying biological activity resulting from a given protein-substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate inhibitor may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein may be provided which adds a domain that permits the protein to be bound to an insoluble matrix.
  • protein-protein interaction component fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • a potential interacting protein e.g., an 3 5 s-labeled polypeptide
  • the test compound and incubated under conditions conducive to complex formation e.g., at 4°C in a buffer of 2mM Tris-HCl (pH 8), InM EDTA, 0.5% Nonidet P-40, and lOOmM NaCl.
  • the beads are washed to remove any unbound interacting protein, and the matrix bead-bound radiolabel determined directly (e.g., beads placed in scintillant), or in the supernatant after the complexes are dissociated, e.g., when microtitre plate is used.
  • the complexes may be dissociated from the matrix, separated by SDS-PAGE, and the level of interacting polypeptide found in the matrix-bound fraction quantitated from the gel using standard electrophoretic techniques.
  • Another aspect of the present invention provides methods for screening various compounds for their ability to inhibit or reverse the progression of tuberculosis. Such methods are referred to within this section as in vivo as they involve the use of whole cells in culture or the use of animals or samples taken therefrom.
  • the subject progenitor cells, and their progeny can be used to screen various compounds.
  • Such cells can be maintained in minimal culture media for extended periods of time (e.g., for 7-21 days or longer) and can be contacted with any compound, to determine the effect of such compound on one of LDH release, cell permeation and/or necrosis, cell growth, proliferation, and differentiation in the culture.
  • Detection and quantification of such properties in response to a given compound provides a means for determining the compound's efficacy at inducing one of the growth, proliferation or differentiation in a given ductal explant.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • Identification of the progenitor cell population(s) amplified in response to a given test compound can be carried out according to such phenotyping as described above.
  • the protein-protein interaction of interest is generated in whole cells, taking advantage of cell culture techniques to support the subject assay.
  • the protein-protein interaction of interest may be constituted in a eukaryotic cell culture system, including mammalian and yeast cells.
  • Advantages to generating the subject assay in an intact cell include the ability to detect inhibitors which are functional in an environment more closely approximating that which therapeutic use of the inhibitor would require, including the ability of the agent to gain entry into the cell.
  • Furthe ⁇ nore certain of the in vivo embodiments of the assay, such as examples given below, are amenable to high through-put analysis of candidate agents.
  • the components of the protein-protein interaction of interest may be endogenous to the cell selected to support the assay. Alternatively, some or all of the components may be derived from exogenous sources. For instance, fusion proteins may be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein. The cell is ultimately manipulated after incubation with a candidate inhibitor in order to facilitate detection of a protein-protein interaction-mediated signaling event (e.g., modulation of a post-translational modification of a protein-protein interaction component substrate, such as phosphorylation, modulation of transcription of a gene in response to cell signaling, etc.).
  • a protein-protein interaction-mediated signaling event e.g., modulation of a post-translational modification of a protein-protein interaction component substrate, such as phosphorylation, modulation of transcription of a gene in response to cell signaling, etc.
  • the effectiveness of a candidate inhibitor may be assessed by measuring direct characteristics of the protein-protein interaction component polypeptide, such as shifts in molecular weight by electrophoretic means or detection in a binding assay.
  • the cell will typically be lysed at the end of incubation with the candidate agent, and the lysate manipulated in a detection step in much the same manner as might be the reconstituted protein mixture or lysate, e.g., described above.
  • Indirect measurement of protein-protein interaction may also be accomplished by detecting a biological activity associated with a protein-protein interaction component that is modulated by a protein-protein interaction mediated signaling event.
  • fusion proteins comprising a protein-protein interaction component polypeptide and an enzymatic activity are representative embodiments of the subject assay in which the detection means relies on indirect measurement of a protein-protein interaction component polypeptide by quantitating an associated enzymatic activity.
  • the biological activity of a nucleic acid-protein, protein- substrate or protein-protein interaction component polypeptide may be assessed by monitoring changes in the phenotype of the targeted cell.
  • the detection means may include a reporter gene construct which includes a transcriptional regulatory element that is dependent in some form on the level of an interaction component or a interaction component substrate.
  • the protein interaction component may be provided as a fusion protein with a domain which binds to a DNA element of the reporter gene construct.
  • the added domain of the fusion protein may be one which, through its DNA-binding ability, increases or decreases transcription of the reporter gene. Whichever the case may be, its presence in the fusion protein renders it responsive to the protein-protein interaction- mediated signaling pathway. Accordingly, the level of expression of the reporter gene will vary with the level of expression of the protein interaction component.
  • the protein-protein interaction component or potential interacting polypeptide may be used to generate an two-hybrid or interaction trap assay for subsequently detecting agents which disrupt binding of the interaction components to one another.
  • the method comprises the use of chimeric genes which express hybrid proteins.
  • a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator may be fused in frame to the coding sequence for a "bait" protein, e.g., a protein-protein interaction component polypeptide of sufficient length to bind to a potential interacting protein.
  • the second hybrid protein encodes a transcriptional activation domain fused in frame to a gene encoding a "fish" protein, e.g., a potential interacting protein of sufficient length to interact with the protein- protein interaction component polypeptide portion of the bait fusion protein.
  • bait and fish proteins are able to interact, e.g., form a protein-protein interaction component complex, they bring into close proximity the two domains of the transcriptional activator. This proximity causes transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene may be detected and used to score for the interaction of the bait and fish proteins.
  • the method includes providing a host cell, such as a yeast cell.
  • the host cell contains a reporter gene having a binding site for the DNA-binding domain of a transcriptional activator used in the bait protein, such that the reporter gene expresses a detectable gene product when the gene is transcriptionally activated.
  • the first chimeric gene may be present in a chromosome of the host cell, or as part of an expression vector.
  • the host cell also contains a first chimeric gene which is capable of being expressed in the host cell.
  • the gene encodes a chimeric protein, which comprises (i) a DNA-binding domain that recognizes the responsive element on the reporter gene in the host cell, and (ii) a bait protein, such as a protein-protein interaction component polypeptide sequence.
  • a second chimeric gene is also provided which is capable of being expressed in the host cell, and encodes the "fish" fusion protein.
  • both the first and the second chimeric genes are introduced into the host cell in the fo ⁇ n of plasmids.
  • the first chimeric gene is present in a chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid.
  • the DNA-binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein are derived from transcriptional activators having separable DNA-binding and transcriptional activation domains.
  • these separate DNA-binding and transcriptional activation domains are known to be found in the yeast GAL4 protein, and are known to be found in the yeast GCN4 and ADR1 proteins.
  • Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful for the present invention, and include, for example, the LexA and VP16 proteins.
  • DNA-binding domains may be used in the subject constructs; such as domains of ACE1, ⁇ cl, lac repressor, jun or fos.
  • the DNA-binding domain and the transcriptional activation domain may be from different proteins.
  • LexA DNA binding domain provides certain advantages. For example, in yeast, the LexA moiety contains no activation function and has no known effect on transcription of yeast genes. In addition, use of LexA allows control over the sensitivity of the assay to the level of interaction. In certain embodiments, any enzymatic activity associated with the bait or fish proteins is inactivated, e.g., dominant negative or other mutants of a protein-protein interaction component may be used.
  • the protein-protein interaction component- mediated interaction if any, between the bait and fish fusion proteins in the host cell, therefore, causes the activation domain to activate transcription of the reporter gene.
  • the method is carried out by introducing the first chimeric gene and the second chimeric gene into the host cell, and subjecting that cell to conditions under which the bait and fish fusion proteins and are expressed in sufficient quantity for the reporter gene to be activated.
  • the formation of a protein-protein interaction component/interacting protein complex results in a detectable signal produced by the expression of the reporter gene. Accordingly, the level of formation of a complex in the presence of a test compound and in the absence of the test compound may be evaluated by detecting the level of expression of the reporter gene in each case.
  • reporter constructs may be used in accord with the methods of the invention and include, for example, reporter genes which produce such detectable signals as selected from the group consisting of an enzymatic signal, a fluorescent signal, a phosphorescent signal and drug resistance.
  • the reporter gene product may be a detectable label, such as luciferase, ⁇ -lactamase or ⁇ -galactosidase, and is produced in the intact cell.
  • the label may be measured in a subsequent lysate of the cell.
  • the lysis step may be avoided, and providing a step of lysing the cell to measure the label will typically only be employed where detection of the label cannot be accomplished in whole cells.
  • the reporter gene construct may provide, upon expression, a selectable marker.
  • a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Reporter genes include those that are readily detectable.
  • the reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties.
  • the product of the reporter gene may be an enzyme which confers resistance to antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (e.g., thymidine kinase or dihydrofolate reductase).
  • aminoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo may be placed under transcriptional control of a promoter element responsive to the level of a protein-protein interaction component polypeptide present in the cell.
  • Such embodiments of the subject assay are particularly amenable to high throughput analysis in that proliferation of the cell may provide a simple measure of inhibition of an interaction.
  • Reporter genes further include, but are not limited to CAT (chloramphenicol acetyl transferase) luciferase, and other enzyme detection systems, such as ⁇ -galactosidase, ⁇ - lactamase, human placental secreted alkaline phosphatase.
  • CAT chloramphenicol acetyl transferase
  • the amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable.
  • specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain, western blots or an intrinsic activity.
  • the product of the reporter gene is detected by an intrinsic activity associated with that product.
  • the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.
  • the amount of expression from the reporter gene is then compared to the amount of expression in either the same cell in the absence of the test compound or it may be compared with the amount of transcription in a substantially identical cell that lacks a component of the protein-protein interaction of interest.
  • assays may be conducted to identify compounds that modulate the activity (e.g. expression) of a gene.
  • the ability of a compound to up- or down-regulate the level of expression of at least one gene is determined by phenotypic analysis of the cell, in particular by determining whether the cell adopts a necrosis-deficient phenotype.
  • the level of expression of a gene is determined, e.g., by using a microarray having probes to the one or more genes. Modulating the expression of a gene in a cell can be achieved, e.g., by contacting the cell with an agent that increases the level of expression of the gene or the activity of the polypeptide encoded by the gene.
  • an agent which modulates the expression of a gene of interest is identified by contacting cells expressing the gene with test compounds, and monitoring the level of expression of the gene.
  • compounds which modulate the expression of a gene can be identified by conducting assays using the promoter region of a gene and screening for compounds which modify binding of proteins to the promoter region.
  • the nucleotide sequence of the promoter may be described in a publication or available in GenBank.
  • the promoter region of the gene can be isolated, e.g., by screening a genomic library with a probe corresponding to the gene. Such methods are known in the art.
  • M. tuberculosis cell that can be obtained from a subject and, e.g., established as a primary cell culture.
  • the cell can be immortalized by methods known in the art.
  • it may be to confirm that the gene expression profile of the cell line corresponds essentially to that of a normal M. tuberculosis cell. This can be done as described in details herein.
  • microarrays may be used in these embodiments, various other methods of detection of gene expression are available.
  • the first step of the methods includes isolation of mRNA from cells, this step may be conducted as described above. Labeling of one or more nucleic acids may be performed as described above.
  • mRNA obtained form a sample is reverse transcribed into a first cDNA strand and subjected to PCR, e.g., RT-PCR.
  • PCR e.g., RT-PCR.
  • House keeping genes, or other genes whose expression does not vary can be used as internal controls and controls across experiments.
  • the amplified products can be separated by electrophoresis and detected.
  • the amplified samples can also be separated on a agarose or polyacrylamide gel, transferred onto a filter, and the filter hybridized with a probe specific for the gene of interest. Numerous samples can be analyzed simultaneously by conducting parallel PCR amplification, e.g., by multiplex PCR.
  • a quantitative PCR technique that can be used is based on the use of TaqManTM probes, which are commercially available, as are protocols for their use. PCR reactions may be set up using the PE Applied Biosystem TaqMan PCR Core Reagent Kit according to the instructions supplied. In another embodiment, mRNA levels is determined by dotblot analysis and related methods.
  • Assay formats which approximate such conditions as formation of protein complexes or protein-nucleic acid complexes, and enzymatic activity may be generated in many different forms, as those skilled in the art will appreciate based on the present description and include but are not limited to assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Assaying binding resulting from a given targetimolecule interaction may be accomplished in any vessel suitable for containing the reactants.
  • Examples include microtitre plates, test tubes, and micro-centrifuge tubes. Any of the assays may be provided in kit format and may be automated. Many of the following particularized assays rely on general principles, such as blockage or prevention of fusion, that may apply to other particular assays. 3. In vivo Assays
  • Animal models of tuberculosis infection and/or disease may be used as an in vivo assay for evaluating the effectiveness of a potential drug target in treating or preventing flavivirus related diseases or disorders.
  • a number of suitable animal models are described briefly below in the Exemplification, however, these models are only examples and modifications, or completely different animal models, may be used in accord with the methods of the invention.
  • Animal models may be developed by methods known in the art, for example, by infecting an animal with M. tuberculosis, or by genetically engineering an animal to be predisposed to such infection (see, e.g., Wu, S.-J.L. et al. Evaluation of the severe combined immunodeficient (SCID) mouse as an animal model for dengue viral infection. Am. J. Trop. Med. Hyg. 52, 468-476 (1995)).
  • SCID severe combined immunodeficient
  • in vivo models are available and may be used when appropriate for specific pathogens or specific test agents. It is also relevant to note that the species of animal used for an infection model, and the specific genetic make-up of that animal, may contribute to the effective evaluation of the effects of a particular test agent. For example, immuno-incompetent animals may, in some instances, be preferable to immuno-competent animals. For example, the action of a competent immune system may, to some degree, mask the effects of the test agent as compared to a similar infection in an immuno-incompetent animal. In addition, many opportunistic infections, in fact, occur in immuno-compromised patients, so modeling an infection in a similar immunological environment is appropriate. 4. Compositions Comprising Modulators and Their Use in Methods of
  • inhibitors, modulators of the subject polypeptides, or biological complexes containing them may be used in the manufacture of a medicament for any number of uses, including, for example, treating any disease or other treatable condition of a patient (including humans and animals), and particularly a disease caused by M. tuberculosis.
  • compositions of this invention include any modulator identified according to the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Methods of making and using such pharmaceutical compositions are also included in the invention.
  • the pharmaceutical compositions of the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, infra articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between about 0.5 and about 75 mg/kg body weight per day of the modulators described herein are useful for the prevention and treatment of disease and conditions, including diseases and conditions mediated by pathogenic species of origin for the polypeptides of the invention.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5%, to about 95%> active compound (w/w). Alternatively, such preparations contain from about 20%> to about 80%> active compound.
  • kits including the subject bacterial strains, nucleic acids, polypeptides, antibodies, and other subject materials, and optionally instructions for their use.
  • Kits comprising the pharmaceutical compositions of the present invention are also within the scope of the invention.
  • the compositions may be pharmaceutical compositions comprising a pharmaceutically acceptable excipient.
  • this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.
  • Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications. EXEMPLIFICATION
  • EXAMPLE 1 Characterization of wild type M. tuberculosis cytotoxicity of lung epithelial cells. While the role for the inhibition of apoptosis in macrophages by virulent M. tuberculosis may be' an important phenomenon preventing the host cell mediated killing of infected cells, virulent M. tuberculosis are known to be significantly cytotoxic to macrophages, fibroblast, and epithelial cells in vitro. We have quantitatively measured the contribution of apoptosis and necrosis to the cytotoxicity of infected epithelial cells (A549), and found that unlike that observed during human macrophage infection, virulent M.
  • tuberculosis induce necrosis not apoptosis of human lung epithelial cells as the primary mode of cell death (data not shown).
  • Necrosis was not induced by exposure of the epithelial cells to the avirulent mycobacteria M. bovis BCG and M. smegmatis, even though M. bovis BCG was found to infect and grow within the epithelial cells, further supporting the previously reported hypothesis that cytotoxicity of these cells is a virulence-associated phenotype of M. tuberculosis.
  • tuberculosis and not by gamma irradiated, heat killed bacilli, or bacterial components found in the whole cell lysates or culture filtrate proteins (CFP).
  • CFP culture filtrate proteins
  • Necrosis and cell membrane disruption of lung epithelial cells could be important to the tissue destruction observed during tuberculosis. Necrosis may also play an integral part in the hematogenous spread of the bacilli as a mechanism of escape from the granuloma (early or reactivation disease) or the bronchial epithelium during the cavitary stage of the disease, leading to transmission of the bacilli. Likewise, this mode of cell death could be important to intracellular survival of the tubercle bacilli and killing of human macrophages in a manner similar to S b ⁇ /zet7 ⁇ -induced cell death of macrophages by a caspase- 1- dependent necrosis. Factors produced by M.
  • tuberculosis such as those responsible for membrane permeation, could also alter phagosomal membranes during infection of macrophages, thereby effecting the endocytic pathway of M. tuberculosis phagosomes or could be involved in facilitating delivery of bacterial-derived effector molecules to the host cell cytoplasm, as suggested for Legionella pneumophila infection of human macrophages and Salmonella enterica.
  • EXAMPLE 2 ⁇ V5367 transposon library construction in M. tuberculosis strain Erdman
  • transposon mutagenesis was determined to be the most efficient method of identifying these factors for further study in this model.
  • the temperature-sensitive phagemid, pJSC84 was amplified in M. smegmatis strain MC 2 155 to produce tittered phage particles.
  • This phage contains the IS 1096 derived transposon, Tn5367, and is a conditionally replicating TM4-based phage that replicates/lyses mycobacteria at 30°C, but does not replicate and lyse the bacterial cells at growth temperatures of 37°C.
  • These phage particles were then used to infect the wild type M. tuberculosis strain Erdman to deliver the transposon according to the methods of Bardorov et al, (1997). Briefly, log phase-grown strain Erdman (O.D.
  • MP buffer 50mM Tris pH 7.6, 150mM NaCl, lOmM MgCl 2 , 2mM CaCl 2 .
  • pre-warmed phage adsorption- stop buffer 50 ⁇ l was added to stop the phage infection and the bacilli were plated on pre-warmed Middlebrook 7H10 plates supplemented with 0.1% Tween 80, 0.4% Casitone, 40 ⁇ g/ml L- tryptophan, 10% OADC, 0.5% Glycerol, 0.5% Dextrose (7H10/TP) containing 50 ⁇ g/ml hygromycin B.
  • each 96-well plate was left empty for transfer of the wild type Erdman positive controls.
  • Eight individual colonies of the wild type Erdman were picked from different plates and integrated into one column of each 96 well plate to serve as positive controls. This allowed for replicate transfer of the transposon library with positive controls by multi-pipette for screening of this library. Transfer of these hygromycin resistant recombinants with the eight wells of wild type Erdman was continued until each batch of the generated mutants was tested for LDH activity as described below.
  • EXAMPLE 3 Screening the transposon library for the ability to induce lactate dehydrogenase (LDH) release from infected A549 cells.
  • LDH lactate dehydrogenase
  • tuberculosis Erdman required to induce LDH release after colonies were transferred and grown in the 96-well plates , 2.) the amount of A549 cells required to produce the least amount of background LDH release in the 96-well format, and 3.) the time point for significant LDH release detection.
  • the first batch of recombinants (680) in the transposon library was then screened for the ability to induce LDH release from infected A549 cells. Briefly, A549s were laid down at lxl0 5 cells/ml, 200 ⁇ l/well in a 96 well flat bottom plate with 1%FBS/MEM. Cells were incubated for 48 h in 5%> CO 2 at 37°C. Prior to infection, each well of a 96-well plate containing the ⁇ hygromycin resistant recombinants and the wild type M. tuberculosis Erdman control, as described above, was monitored visually to ensure sufficient growth had occurred in each well.
  • % cell permeation [(Release of LDH from infected cells- Background release)/(Maximum release of LDH-Background release)]* 100.
  • Figure 1 shows a typical result of 64 hygromycin resistant recombinants and eight wild type Erdman M. tuberculosis transfers in one 96-well plate assay. Although the majority of the wild type Erdman transfers ranged from 12-16%
  • Partial Sau3A digests were performed on chromosomal DNA from each mutant, by diluting the enzyme in a 10-fold dilution series and incubating at 37°C for 30 min to 1 hour.
  • Rv3874::TN5370 had an insertion into the Rv3874 gene.
  • both genes have unknown functions although some homology to other known proteins and characterizations exist (Table 1).
  • the Rv3874::TN5370 gene is located in the RDl region of the M. tubei-culosis chromosome; a region associated with loss of virulence in the attenuated strains of M. bovis
  • M. bovis BCG (Maheiras et al., 1996). Rv3874 is transcribed together with the ESAT-6 gene product in M. tuberculosis. We also showed previously that M. bovis BCG did not have the LDH and necrosis phenotype in the A549 cell models, thus further supporting the role of RDl in the virulence of both M. tuberculosis and M. bovis.
  • the M. tuberculosis Erdman nee mutants were complemented with an integrative plasmid containing each respective gene or control plasmid to determine if the gene restored the parental phenotype. Because the Rv3874 gene is in an operon with
  • Rv3875 the complete operon was first used for complementation. As the transposon in Rv3874 may be affecting Rv3875 by polar disruption, both genes will also need to be complemented individually (i.e. by knocking out the other gene in the complementing operon) as described in below.
  • the Rv3874/Rv3875 operon was first amplified from M. tuberculosis Erdman genomic DNA using primers surrounding the operon, with Hindlll sites on each 5' end (forward 5' aagcttagctcccgtaatgacaac and reverse 5' aagcttacgaactcggcgttgccctat).
  • the amplified operon was then be ligated into the vector pCR2.1 and transformed into TOP 10 'F cells (Invitrogen), according to the manufacturers instructions. To confirm the nature of the insert, sequencing was performed as described above.
  • the inserts in the pCR2.1 vector were then excised from the pCR2.1 vector using the Hindlll sites at either end of the insert, gel purified using the Qiaex gel extraction kit (Qiagen) and ligated into the Hindlll digested integrating vector pMV306 kan , using T4 DNA ligase.
  • mice 15 SCID mice per treatment were consecutively placed into restraining devices and injected iv, into the tail vein, with 2 x 10 6 bacilli (CFU counts on Middlebrook 7H10 medium were performed to confirm the initial dose; Figure 7A).
  • Spleen, liver and lungs of 5 mice per treatment were harvested after 24 hours of infection and the number of bacilli per organ was determined using CFU counts on appropriate Middlebrook 7H10 media ( Figure 7B), following homogenization. The mice were then observed for signs of infection regularly and time of death was recorded for the remaining 10 infected mice for each treatment.
  • bovis BCG may be the cause of its attenuation of necrosis and virulence in mice. Therefore, we must now focus our studies on the complete characterization of these nee genes in both in vitro and in vivo models, determine the mechanisms of host cell necrosis and the relationship to lung pathology, the role of each nee gene in this phenotype, and determine when the necrosis phenotype is involved in the pathogenesis of tuberculosis.
  • EXAMPLE 5 Characterization of the defect in Rv3874:: ⁇ N5370 and
  • the gene responsible for loss of the necrosis phenotype may be identified as the transposon disrupted gene as follows. Following identification, an integrating vector, such as for example, pMV306 kan , may be used to restore the functional gene in the transposon- disrupted strains. This will restore the parental phenotype to the mutant strain, thus fulfilling Koch's molecular postulates. As the transposon disrupted mutant strains may potentially revert, non-revertible mutants may be constructed using homologous recombination. These stable mutants and wild type M.
  • an integrating vector such as for example, pMV306 kan
  • tuberculosis may then be studied in a mouse model of tuberculosis to determine the role of host cell necrosis to the growth, tissue pathology, hematogenous spread, and virulence in mice.
  • mutants in this category may be tested for virulence in the SCID mouse aerosol model.
  • SCID mice may be infected with each mutant, complemented mutant, and wild type M. tuberculosis Erdman using the low dose aerosol infection model (Schwebach et al., 2002) to study time of survival, dissemination into the liver and spleens, and lung histopathology (as described below). Because the inoculum deposition could have an effect on the course of M.
  • tuberculosis infection and disease we use a well-defined aerosol infection model that deposits only a few bacteria into the upper airways to mimic more natural M. tuberculosis infection (Schwabach et al., 2002). All mutant strains, complemented mutant strains and wild type M. tuberculosis Erdman control bacilli may be confirmed in the LDH release assay and necrosis assay, as previously described.
  • the mouse tuberculosis model offers numerous advantages, including the close correlation of pathology to human tuberculosis, availability of numerous "knockout" strains of mice to study host immunopathology, and numerous reagents such as monoclonal antibodies to cytokines and chemokines, T cell and macrophage cell lines, and reagents for the detection of cellular activation cascades etc. for correlation analysis of intracellular survival, growth, and killing caused by M. tuberculosis.
  • Our strategy is analysis of each necrosis mutant compared to wild type in the immune incompetent SCID mouse model for virulence. This model is more sensitive to killing than immune competent mice, provides study of M. tuberculosis in a model with reduced cell-mediated immunopathogenesis, and is necessary to detect different degrees of attenuation of virulent M. tuberculosis.
  • the mutant strains, complemented mutant strains and wild type M. tuberculosis Erdman will then be tested for growth, tissue pathology, hematogenous spread and virulence in the immunocompetent Balb/c mouse strain by the low dose aerosol infection methods by the collaborating laboratory (Schwebach et al., 2002).
  • Balb/c mice may be used for pathology, growth, and virulence studies. These mice are immune competent and may be used to compare the growth, pathology and virulence of M. tuberculosis wild type and mutants to the SCID mice infections, but in the 'presence of host immunopathogenesis.
  • mice for experiments with either mouse model, all mutant strains are grown to an O.D.600 nm 1 in 7H9 media plus OADC, glycerol and Tween with or without antibiotics. The cultures are washed twice in an equal volume of PBS/Tween buffer, then resuspended in 0.05% Tween with 0.4% Antifoam-A PBS buffer. The bacterial suspension is sonicated to reduce clumping. 12 mice per treatment, may be infected, as previously described in Schwebach et al., 2002 with ⁇ 100 bacilli. (CFU counts will also be carried out to confirm the initial dose by plating out on 7H10/OADC plates from the isolated lungs of 2 mice each treatment).
  • inoculating methods are based on the nebulizer doses of bacilli required to achieve a low dose infection by plating CFU from sentinel mice from lungs three hours post infection. 2 mice are sacrificed per treatment, at each time point of day 1, 7, 21, 42 and 84. Samples of liver, lung and spleen may be taken for pathology, and the remainder of the tissue sample may be homogenized, and then plated out on 7H10/OADC plates for CFU determination to determine the level of hematogenous spread. b. Completion of the generation of the complementing clones to Rv3874 and Rv3351c and testing if these complementing clones restore the parental phenotypes to the mutants in both the in vitro and SCID mouse models.
  • the M. tuberculosis Erdman nee mutants have been complemented with genomic regions containing each respective gene to quickly determine if the mutation in each mutant was specifically related to the virulence of M. tuberculosis and as a means to begin to satisfy Koch's molecular postulates for each gene (see section c).
  • the Rv3874 gene is in an operon with Rv3875 (Berthet et al.., 1998a) and the transposon in Rv3874 may be affecting Rv3875 by polar disruption, both genes will also be complemented individually (i.e. by knocking out the other gene in the complementing operon).
  • the Rv3874/Rv3875 operon has been amplified from M.
  • the pCR2.1 vector plus the whole operon insert may be digested with S ⁇ tJ and SexAl (for which there are complementing sites in the middle of Rv3874 only), then digested with Klenow DNA polymerase I for 20 min at room temperature, with the addition of 33 ⁇ M each dNTP.
  • the vector may then be re-ligated using T4 DNA ligase and sequenced to confirm the deletion is in frame as described above.
  • the inserts in the pCR2.1 vector may then be excised from the pCR2.1 vector using the Hindlll sites at either end of the insert, gel purified using the Qiaex gel extraction kit (Qiagen) and ligated into the Hindlll digested integrating vector ⁇ MV306 an , using T4 DNA ligase.
  • the purified whole operon PCR product may be digested with Tsp451 (as there are Tsp451 sites within the pCR2.1 vector) and the 508bp fragment, consisting of the whole operon minus the last 60bp of Rv3875, may be gel purified as previously described.
  • the fragment may then be Klenow treated as above to modify the ends, and then ligated into the Klenow treated Xbal digested pMV306 an vector.
  • the vector, plus insert may then be sequenced using the T7 and SP6 sequencing primers.
  • the clones for use in complementation of the Rv3874/Rv3875 operon are shown in Table 2. All constructs may then be transformed into the nee A mutant (Ollar and Coimell, 1998; Hatfull and Jacobs, 2000). In cases where nee genes can not be complemented with the integrative plasmid, non-revertible deletions of these genes in other M. tuberculosis may be generated to confirm the role of the nee gene to the phenotype.
  • pMV306 kan is an integrating vector with a kanamycin resistance gene.
  • Small in-frame chromosomal deletions of selected genes may be made in the wild type M. tuberculosis strain Erdman or other strains using transduction methods. Double- gene knockout mutants of necA and necB (or other nee genes important to the cellular necrosis and virulence of M. tuberculosis) may also be constructed. A number of innovative strategies have been used with varying degrees of success to generate knockout mutants of M. tuberculosis. Until recently, each of these approaches had major drawbacks and were not easily adapted by other laboratories (Pelicic et al., 1996, 1997; Azad et al.,1997, Pavelka and Jacobs, 1999).
  • allelic exchange a novel method for the generation of targeted deletion mutations by allelic exchange was developed using in vttro-generated specialized transducing mycobacteriophages (Bardorov et al., 2002) that has been successfully transferred to other laboratories with equal success.
  • the utility and reproducibility of the method was demonstrated by the construction of three different auxotrophic mutant strains of M. tuberculosis by Bardorov et al., 2002.
  • This allelic exchange method may be used to interrupt nee ORF's (and other identified ORF's important to the necrosis phenotype).
  • tuberculosis genome sequence database at http://genolist.pasteur.fr/TubercuList) that give the maximal amount of DNA flanking both sides of the M. tuberculosis ORF.
  • These DNA fragments may be cloned into the pBluescript KSII cloning vector (Stratagene) and deletion methods performed to generate allelic exchange substrates (AES).
  • AES allelic exchange substrates
  • the resultant inserts continuing ORF's with deletions may then be cloned into specific cosmids and then subcloned into the Pad site of the specialized transducing phage phAE87 DNA after restriction digestion of the insert and vector.
  • PhAE87 may be packaged and transduction performed as described by Bardorov et al., 2002.
  • Each deletion mutant is confirmed by sequencing of the PCR fragments from genomic DNA and then tested in the in vitro and in vivo models described below. d. Testing the level of expression of necA and necB genes by in situ
  • M. tuberculosis causes a progressive disease that begins with infection that develops into cavitary disease in both animal models and during human disease. Likewise, these phenotypes are known to be influenced by the host immune response to infection. At which stage of infection the necA, necB, and other putative nee genes are expressed in the aerosol infection Balb/c mouse model may be determined as follows. In situ hybridization has been successfully used to locate both M. tuberculosis DNA and mRNA in human lung specimens (Fenhalls et al., 2002a/b).
  • the protocol of Fenhalls el al., 2002 may be used to paraffin-embed AFB-positive mouse lung tissues collected from Balb/c mice from early low dose infection (2 hours) and over the course of disease at each time point of day 1, 7, 21, 42 and 84.
  • Control tissues may also be collected from uninfected mice.
  • Paraffin embedded tissues and control M. tuberculosis Erdman embedded cells (from Middlebrook 7H9 broth grown cultures) may be be cut into 5-um sections using a microtome, and applied to RNase-free slides prepared coated with 5 ⁇ g/ml aminopropyl-triethoxy-silane (Sigma Aldrich). Positive confirmation for the presence of M. tuberculosis in tissues may be made by AFB staining.
  • Duplicate slides may be made from each tissue section and one slide used for AFB staining after dewaxing to determine the presence of M. tuberculosis in that tissue section.
  • the second slide may be probed with nee gene riboprobes and a M. tuberculosis DNA probe to determine the presence M. tuberculosis and nee gene transcripts in each tissue specimen.
  • the M. tuberculosis specific DNA probe may be constructed from PCR amplification of the rpoB gene from M. tuberculosis Erdman genomic DNA (Rv0667), purified, and stored until needed for the in situ detection of M.
  • RNA probes may be constructed from DNA fragments that are PCR amplified regions of complete nee genes from M. tuberculosis genomic DNA.
  • the fragments may be purified and cloned into the vector pGEMTeasy (Promega) with flanking T7 and SP6 ribosomal binding sites.
  • Antisense and sense biotin-labeled nee gene riboprobes may then be made by transcribing the nee genes using T7 with SP6 RNA polymerases (Gibco BRL, Switzerland).
  • Each probe may be tested for biotinylation by spotting the probes onto nitrocellulose membranes and detected with streptavidin-conjugated alkaline phosphatase with NBT/BCIP/INT substrate (Roche).
  • streptavidin-conjugated alkaline phosphatase with NBT/BCIP/INT substrate (Roche).
  • Negative detection of nee mRNA from control uninfected lung tissue embedded cells and for positive detection of nee gene transcripts from M. tuberculosis embedded cells from broth grown cultures may first be performed. Each tissue section and hybridization mixture containing the nee riboprobe will first be heated at 95°C and placed onto ice for one minute.
  • Tissue sections on slides may then be incubated with the hybridization mixture for 18 hours at 50°C in a CO2 incubator and nee genes detected with streptavidin-conjugated alkaline phosphatase with NBT/BCIP/INT (Gibco). Tissues may then be counter-stained with haematoxylin (Vector, USA) for 10 seconds, rinsed in distilled water, mounted with permamount, and slides viewed with a Zeiss microscope to score the presence of nee gene transcripts (brown color).
  • Anti-murine- CD14 antibodies (to identify murine macrophage-like cells; Becton Dickinson) and cytokeratin staining (to identify epithelial cells) and osmium tetroxide/tannic acid staining (to identify lamellar bodies of murine pneumocytes (Corti et al., 1996 ) may be used in co-localization studies of nee DNA and mRNA expression.
  • Riboprobes may be detected on separate AFB-positive lung tissues as described above, and then cytokeratin and tetroxide/tannic acid staining and Mab's may be tested on AFB-positive lung tissues as described by Fenhalls et al., 2002c.
  • Nonspecific proteins may be blocked with 5% milk powder in PBS in 0.5% Triton-X-100 for 30 min at room temperature.
  • the slides may then be incubated with each pneumocyte stain or anti-murine-CD14 antibodies (after testing the titer required to detect control macrophages in lung tissues).
  • slides will be additionally washed three times in PBS, incubated for one hour with secondary antibody (biotinylated/goat anti-mouse; Becton Dickinson), and label detected using streptavidin conjugated alkaline phosphatase (Vector Laboratories, Burlingame, CA) for 30 minutes.
  • Slides may then be washed in PBS and incubated with a solution of fast red (Vector labs) for 30 minutes.
  • the slides may be viewed pre and post counter-staining as described above to determine which method best discerns the detection of macrophages with both M. tuberculosis DNA and mRNA. e. Testing the interaction of the gene products of the necA/Rv3875 operon independently and with necB using a standard yeast two-hybrid system.
  • necA and necB genes generated a loss of host cell permeation of epithelial cells (although at different degrees) but a different virulence phenotype upon intravenous infection of SCID mice
  • whether the necA gene product or ESAT-6 interact with the necB gene product may be determined using the CytoTrap two-hybrid system (Stratagene).
  • the interaction of these bait molecules with a M. tuberculosis library may be studied to determine if these nee genes interact with other gene products of M. tuberculosis using methods developed by Steyn et al., 2002 but with the CytoTrap system.
  • tuberculosis into the cytosol presumably from within a vacuole of an infected cells
  • this unique system may identify protein-protein interactions that require the environment of the cytosol or a plasma membrane for proper interaction.
  • the CytoTrap system contains the yeast strain cdc25H, which harbors a temperature-sensitive mutation in the cdc25 gene (the yeast homologue for hSos) and thus yeast cells cannot grow at 37°C.
  • Two unique vectors are used, the pMyr vector for the target protein and pSos for the bait protein. This system allows for the determination of cloned protein-protein interactions by activation of the Ras pathway in yeast (due to the recruitment of the human Sos gene product (hSos) to the membrane of the cells) to overcome temperature restriction (caused be mutation of the cdc25 protein) and growth of positive recombinants at 37°C.
  • This system may be used to confi ⁇ n the interaction of the necA and Rv3875 gene products (as shown biochemically by Renshaw et al., 2002) by cloning the necA gene (target) into the MCS of pMry (which creates a fusion protein with a myristylation sequence that anchors the fusion protein containing the NecA to the yeast plasma membrane) and the Rv3875 gene (bait) into the MCS pSOS (generating a fusion protein with hSos and the Rv3875 protein free in the cytosol of the yeast cells).
  • fusion proteins may then be coexpressed in the cdc25H yeast strain, and the recombinants cultured at the restrictive temperature of 37°C (testing with control plasmids provided by the manufacturer to confirm fusion protein interactions). If the bait (Rv3875) and target (NecA) proteins interact at the cytosolic surface of the plasma membrane, then the hSos fusion protein will also be recruited to the yeast plasma membrane thereby activating the plasma membrane associated Ras-signaling pathway and allowing the yeast cdc25H strain to grow and form colonies.
  • both NecA and Rv3875 may be tested as target proteins in pMry with the NecB in pSos as bait to determine if either of the genes in the RDl operon interact with Nec-5. Interactions of recombinant proteins may be confirmed using the ProFound protein-protein interactions kits (Promega) that allow the detection of proteins using western blotting or sequence analysis of bait proteins bound to an interacting biotinylated protein.
  • EXAMPLE 6 The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue Bacillus Calmette-Guerin (BCG) was first isolated from Mycobacterium bovis after serial subculturing in ox bile medium, when Drs. Calmette and Guerin set out to test the hypothesis that a bovine tubercle bacillus could transmit pulmonary tuberculosis after oral administration. However, unexpectedly after the 39th passage, the strain was unable to kill experimental animals, and showed no reversion to virulence even after the authors had performed over 200 passages, which is consistent with the attenuating mutation being a deletion mutation.
  • BCG Bacillus Calmette-Guerin
  • BCG was determined to be able to protect animals receiving a lethal challenge of virulent tubercle bacilli, and in 1921 was first used as an anti-tuberculous vaccine.
  • an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis, yet the mechanism that causes the attenuation of BCG remains unknown.
  • Mahairas et al. first compared the genomic sequences of BCG and M. bovis, by using subtractive hybridization, and found that there were three regions of difference
  • mice BALB_c and C57BL_6 mice were purchased from Charles River Breeding Laboratories. Severe combined immunodef ⁇ cient (SCID) (BALB_c background) mice were purchased from the National Cancer Institute. Aerosol infections at a low dose of 50 colony-forming units (cfu) per mouse and high dose of 500 cfu per mouse were carried out as described in Schwebach, et al (2002). I.v. injections in the tail vein (at a dose of 2 X 106 cfu) and plating were carried out as in McAdam, et al. (20020. At least 10 mice were used for survival studies, and at least 3 mice per time point for all other studies. For pathology, half of the organ was plated and half was fixed in either 10% formalin (for light microscopy) or 2.5% glutaraldehyde, in 0.1 M sodium cacodylate buffer (for electron microscopy).
  • the unmarked deletion mutant of M. tuberculosis H37Rv, mc24002 was generated by transformation using a sacB counter selection, as described in Pavelka, et al. (1999). Specifically, the plasmid pJH506 was created by cloning UFS (upstream flanking sequence) and DFS (downstream flanking sequence; see Figure 9A) into ,pJH12 [a pMV261 -derived Mycobacterial shuttle plasmid in which UFS and DFS flanked a green fluorescent protein gene (GFPuv, CLONTECH) controlled by the Mycobacterium leprae promoter of 18 kDa].
  • UFS upstream flanking sequence
  • DFS downstream flanking sequence
  • ⁇ JH508 was created by cloning UFS-DFS-GFPuv (from pJH506) into pyUB657. After transformation into mycobacteria, selection was carried out using hygromycin, followed by 3% sucrose. Southern analysis, using UFS- or DFS-specific probes, was performed to confirm the RDl deletion. The mycobacteriophage-based method of specialized transduction, which utilizes conditionally replicating shuttle phasmids, was also used to construct RDl mutants, by using UFS and DFS as above, along with the individual gene constructs (Rv3871, Rv3872_3, Rv3874_5, and Rv3876_7), as described in Bardarov, et al. (2002). Again, Southern analysis was used to confirm deletion.
  • a transposon library of M. tuberculosis Erdman (of «6,460 clones) was constructed as in Cox, et al (1999) by using the hygromycin-resistant Tn5370 transposon. Screening of the library was carried out by infection of A549 cells in 96-well plates (without the addition of gentamicin), which were then screened for reduced lactate dehydrogenase (LDH) release (described in the infection experiment methodology). Identification of the transposon insertion sites was carried out by sequencing as described in Cox, et al.
  • Complementation of Mutants were performed with the cosmid 2F9, which contains the entire RDl region (Rv3860-Rv3885c) in the integration proficient vector pYUB412 (constructed by F. Bange, Medical School, Hannover, Germany, identified and provided by S.T. Cole Institut Pasteur, Paris). After transformation of the mutant strains with the constructs, as described in Bardarov, et al (1997), Southern analyses was carried out to determine whether integration was successful.
  • A549 cells [an alveolar epithelial cell line, American type Culture Collection (ATCC)] were infected at a multiplicity of infection of 10: 1, as described in detail in Dobos, et al 2000b. Briefly, cells were seeded at 5 _ 105 cells_ml, and, after infection with freshly sonicated bacilli for 3 h, any extracellular bacilli were washed off and gentamicin (Sigma) was added to the medium. LDH release was quantified at specified time points by using the cytotoxicity detection kit (Roche Diagnostics), according to the manufacturer's instructions.
  • the maximum and background controls used were 0.1% Triton X-100-treated cells (maximum) and uninfected cells (background). Values were calculated by using the following equation: % LDH release _ (samplebackground) ⁇ (maximum-background).
  • the levels of both apoptosis and necrosis were determined by elucidating the concentration of histone- associated DNA fragments (in the cell lysate and supernatant, respectively), which were quantified by using the cell death detection ELISA (Roche Diagnostics), again according to the manufacturer's instructions.
  • the necrosis positive control was 0.1% Triton X-100, whereas 10 _M campothecin (Sigma) was added for the apoptosis positive control.
  • RDl Deletions of M. bovis andM tuberculosis Are Attenuated for Virulence in Mice.
  • Figure 9A To test whether RDl was essential for virulence in M. bovis and M. tuberculosis, RDl was deleted from the M. tuberculosis strains H37Rv, Erdman, and CDC 1551, and from M. bovis Ravenel ( Figure 9B). The deletion of RDl resulted in the attenuation of virulence of M. tuberculosis H37Rv _RD1 in a SCID mouse model of infection, which was restored on complementation with RDl ( Figure 10A).
  • mice were challenged with 50-200 cfus of the acriflavin-resistant strain of M. tuberculosis Erdman, by the aerosol route. Twenty-eight days after the challenge, the mice were killed, and the bacterial burden in the lungs and spleens was determined (see Table in Figure 11). In mice challenged after 8 months, both the BCG- vaccinated and M.
  • tuberculosis _RD1- vaccinated mice exhibited _ 1 log protection in the lung, with similar cfu values. Protection was seen to a similar extent in mice challenged 3 months after vaccination (see Table in Figure 11). The M. tuberculosis _RD1 mutant also protected against hematogenous spread at both 3- and 8-month challenges (see Table in Figure 11). Thus, M. tuberculosis _RD1 exhibits long-term immunogenicity similar to BCG.
  • M. tuberculosis The cfp-10 esat-6 Operon of M. tuberculosis Is Required for Host Cell Lysis. Because virulent M. tuberculosis, but not BCG, was previously reported to induced lysis of alveolar epithelial cells, we screened for mutants that were defective in the ability to induce cytolysis of lung epithelial cells (Dobos, et al. (2000b; McDonough, et al. (1995)). Approximately 700 clones of a transposon-generated library of M. tuberculosis Erdman mutants were screened for reduced cytolysis of lung epithelial cells, by measuring LDH release.
  • ESAT-6 protein either alone or in combination with culture filtrate protein 10 (CFP-10)
  • CFP-10 culture filtrate protein 10
  • the purified ESAT-6 protein again either alone or in combination with CFP-10, formed structures similar to those seen in amyloidogenic protein solutions before amyloid fibrils are formed (data not shown), and that some amyloidogenic proteins exhibit pore-forming properties similar to those of bacterial toxins.
  • Rv3871 and Rv 876 Rv3877 are Required for Virulence and for the Secretion of ESAT-6.
  • Rv3874 and Rv3875 were the only genes of RDl required for virulence.
  • we generated deletions of the other transcriptional units in RDl by specialized transduction and observed that the deletion of Rv3871 and Rv3876_77, as well as Rv3874_75, resulted in the same attenuated phenotype as the _RD1 or Rv3874::Tn5370 mutants in the SCID mouse model ( Figure 14A).
  • the attenuation by the RDl deletion is not solely the result of the loss of the function of the ESAT-6 protein but also results from loss of the secretion apparatus responsible for the extracellular transportation of ESAT-6.
  • CFP-10 reportedly associates as a chaperone with ESAT-6, which is consistent with a secretion apparatus, and that both of these proteins, which are known to be secreted antigens, do not possess typical signal sequences.
  • a recent report has hypothesized that CFP-10 and ESAT-6 are part of a family of uniquely secreted proteins that may use a novel secretion pathway, and our data support that Rv3871 and Rv3876_ Rv3877 are part of this secretion system.
  • RDl encodes at least three attenuating mutations, all of which participate in the synthesis or secretion of the effector protein ESAT-6. Furthermore, the secreted ESAT-6 protein mediates cytolysis of the infected cell, resulting in increased egress of the bacilli from the cell and an associated increase in tissue invasiveness. Further studies are wareanted to determine the cellular localization, the in vivo expression patterns, and any additional functions mediated by the CFP-10 and ESAT-6 secreted proteins. In addition, whereas our studies show that protection after vaccination with the RDl mutant is not greater than BCG, this may reflect a limitation of the mouse model of infection and warrants further studies in other protection models. This knowledge may lead to the development of novel therapeutic strategies designed to limit tuberculous pathogenesis.
  • Nee genes are believed to cause cell death by induction of cellular activation cascades and/or pore formation of host cells. It is well known that bacterial factors activate signal transduction pathways (e.g. G-protein mediated signal transduction of the pertussis toxin), cleave vesicle-associated membrane proteins that effect intracellular vesicular trafficking (e.g. tetanus toxin), induce pores or disruption of cell membranes effecting the cell permeability barrier (e.g. E. coli hemolysin), and are directly involved in the induction of cell death via apoptosis through induction of host cell enzymes (e.g. Shigella induction of caspases).
  • signal transduction pathways e.g. G-protein mediated signal transduction of the pertussis toxin
  • cleave vesicle-associated membrane proteins that effect intracellular vesicular trafficking e.g. tetanus toxin
  • caspase- 1 is not required for host cell apoptosis, these studies clearly showed that, like other mechanisms of bacterial toxins, primary necrosis can be induced by novel programmed pathway that leads to cell death. Likewise, secondary necrosis may be induced by pore forming molecules that distort the plasma membrane of host cells. Because M. tuberculosis induces plasma membrane distortions that lead to release of LDH early in the infection of epithelial cells (indicative of primary necrosis) but also causes cell cytotoxicity and lysis later during the infection (indicative of secondary necrosis), these two mechanisms will be tested in macrophages and epithelial cells as described below.
  • tuberculosis are only expressed during infection and/or are activated upon expression within the phagosome or upon release into the cytosol.
  • Each selected nee mutant may be tested using these assays to determine if a loss of the specific phenotype occurs as compared to wild type M. tuberculosis and determine the requirement of phagocytosis and intracellular growth rate of each new nee mutant selected for further study in epithelial cells and macrophages.
  • the influence of cellular activation cascades on the membrane permeation (LDH assay), membrane distortion (Glycine protection), and necrosis (cell death ELISA) of infected epithelial cells and macrophages during wild type M. tuberculosis infection may also be determined.
  • the subcellular location of selected nee gene products within wild type M. tuberculosis infected epithelial cells and macrophages may be determined to understand if and then how these gene products escape from M. tuberculosis phagosomes to interact with host cell organelles or membranes.
  • macrophages are the primary host cell for M. tuberculosis infection during disease, phagosomes of infected macrophages are permeable to protein and macrophages also undergo necrosis during the intracellular growth of M. tuberculosis, host cell membrane permeation and/or necrosis by wild type M. tuberculosis could also be important to the intracellular survival and growth of M. tuberculosis in and killing of human macrophages.
  • all mutants found to be defective in the membrane permeation and necrosis of epithelial cells may be studied in comparison with wild type M. tuberculosis using murine and human macrophages for each of the experiments described below.
  • the C57BL/6-derived murine bone marrow macrophage cell line, BMA3.1A7 may be obtained from AECOM and maintained as previously described.
  • Peripheral blood monocytes may be obtained from tuberculin-negative, healthy individuals from the Emory Blood Services Lab and prepared as follows. Blood may be collected in acid citrate dextrose (ACD). 1 ml of 6% Genfran may be added per each 10 ml of blood, mixed gently, and incubated at 37°C for 1-2 h.
  • the upper layer containing the white cell population and some red blood cells may be collected, and the cells washed with Hank's balanced salt solution (HBSS) without Ca ++ or Mg ++ (Gibco) by centrifugation at 1000 x g for 10 min in a variable angle centrifuge.
  • HBSS Hank's balanced salt solution
  • the cell pellets may be resuspended in HBSS (-10 ml per every original 50 ml volume of blood) and overlaid onto 5 ml of Ficoll/Hypaque in 15 ml polypropylene tubes.
  • the suspensions may be centrifuged again at 1000 x g for 30 min, and cells in the interface collected and washed twice with HBSS as above.
  • the cells may be resuspended in IMDM + 10% pooled human serum.
  • the cultures may be incubated at 37°C in 5% CO 2 for 4-5 days and then washed to remove the non-adherent cells to obtain a predominantly monocyte-derived macrophage population.
  • Cellular densities of both the BMA3.1A7 and PBMCs may be adjusted to seed 2 x 10 6 cells per well (2 ml volumes per well). Before each experiment, the number of cells per well may be determined by trypsinizing 6 wells and counting the cells with a hemocytometer. The average of the six wells may be used as the number of macrophages per well for each independent experiment with M. tubercidosis. Adherent macrophages may be infected by removal of media and addition of fresh media (containing wild type or mutant M. tuberculosis) at a multiplicity of infection (MOI) of 100:1, 10:1, and 1:1 as previously described (Mehta et al., 1996; Dobos et al., 2000b).
  • MOI multiplicity of infection
  • Infected and control macrophages may be incubated at 37°C, 5% CO 2 for 6 hr for early infection studies or 1, 3, 5, and 10 days to allow for intracellular replication. At each time point, intracellular CFU, LDH release, and necrosis/apoptosis may be measured as previously described (Dobos et al., 2000b). Transmission electron microscopy may also be used to correlate intracellular numbers and location of mutant and wild type M. tuberculosis.
  • M. tuberculosis uptake of M. tuberculosis is mediated by both microtubule and microfilament dependent pathways, and this adherence and uptake may be mediated by betai integrin and antivitro/zectin receptors.
  • M. avium has been shown to disrupt the macrophage actin filament network during infection perhaps as a means to interfere with vesicle trafficking. To establish whether phagocytosis of M.
  • tuberculosis by epithelial cells and macrophages is a requirement for cell permeation and/or necrosis by the wild type strain Erdman, the microtubule inhibitor cytochalasin D (3 ⁇ M), the microfilament inhibitor colchicine (10 ⁇ M), and the monoclonal antibodies anti-CD29 (betai integrin) and anti-CD51 (vitrowectin receptor) may be used to first determine if adherence and uptake of strain Erdman is also inhibited compared to the previously reported H37Rv strain in A549 cells and macrophages (Bermudez and Goodman, 1996).
  • the adherence and uptake assays may be performed as previously described for counting CFU of intracellular and extracellular bacilli (Be ⁇ nudez and Goodman, 1996; Laochumroonvorapong et al., 1997; Dobos et al., 2000b).These inhibitors may reduce the adherence and uptake of strain Erdman. These experiments may be repeated and monolayers tested for the ability of M. tuberculosis to induce cell permeation (LDH release and ethidium bromide exclusion; see below) and necrosis (cell death ELISA) using the inhibitors. Enumeration of bacilli by light and electron microscopy may also be performed to correlate the numbers of intracellular bacilli. b. Use of confocal microscopy, GFP-labeled bacilli, and Mab's to determine the subcellular location of gene products and trafficking in epithelial cells and macrophages.
  • each identified gene product(s) shown to be involved in membrane permeation and/or necrosis of lung epithelial cells and macrophages may be determined as follows. Infection of the cells by GFP-labled M. tuberculosis may be accomplished as previously described (Teitelbaum et al., 1999b), and localization may be accomplished using a Zeiss LSM410 Laser confocal microscope. This may allow the determination of the location of the gene products or lack thereof from mutant M.
  • tuberculosis at various stages of infection including; interaction with the membranes of phagosomes, release into the cytosol, co-localization with early and late endosomal markers on phagosomes (mannose receptor/Rab5 for early endosomal markers and LAMP-1 /cathepsin D for late endosomal markers), and uptake of various sized fluorescent proteins delivered into the cytosol of cells to determine the size of pores in the M. tuberculosis phagosomes as described above.
  • the pYUB921 shuttle plasmid (described in Teitelbaum et al., 1999b) containing the green fluorescent protein (GFP) of Aqueorea victroia may be used to transform wild type and mutant M. tuberculosis, and expression of GFP in these transformants may be confirmed by fluorescent microscopy.
  • GFP green fluorescent protein
  • the methods for cellular antigen localization using confocal microscopy have been described in detail elsewhere (Griffiths, 1993; Barker et al., 1997; Teitelbaum et al., 1999b). If the gene products identified in this proposal are known antigens of M. tuberculosis, then available monoclonal or polyclonal antibodies (e.g.
  • Mab's to the gene products of necA and Rv3785 that have already been obtained) to these known antigens will be used in subcellular localization studies.
  • the initial focus may start on the NecA/Rv3785 gene products (CPF-10/ESAT-6 which are expressed in the culture filtrate (CFP) of M. tuberculosis) described above, it is anticipated that other genes may be expressed upon infection or intracellular growth of M. tuberculosis that are also important to this cell death phenotype.
  • the gene product(s) is from a gene with no known function (such as the case for necB) or not found to be secreted in the culture supernatant where these antigens could be confirmed by amino acid sequence analysis as previously described (Towbin et al., 1979; Dobos et al., 2000ba), then development of recombinant proteins and generation and testing of polyclonal antisera to the purified recombinant molecules may be required prior to immunocytochemical analysis. In this case, putative restriction enzyme maps for subcloning the intact ORFs for recombinant expression may be developed using the M. tuberculosis genome data base at http://genolist.pasteur.fr/TubercuList: Cole et al., 1998).
  • Data base analysis may also be used to determine if any of the unidentified open reading frames in the database carry homologous mycobacterial signal sequences indicating potential secretion of the gene products (Cole et al., 1998; Tekaia et al., 1999). This information may be useful during recombinant gene expression studies.
  • DNA primers may then be constructed flanking restriction sites in the selected DNA fragments and amplified from M. tuberculosis genomic DNA (Belisle and Sonnenberg, 1998) by PCR using standard techniques. Amplified products may be gel purified and the DNA from the amplified DNA fragments may be restriction mapped to confirm the electronic restriction patterns generated above.
  • Restriction sites in these DNA fragments may be used for cloning these fragments in frame to the T7 promoter of the vector pET30b (Novagen, Inc., Madison, WI) and subcloned into this vector in the E. coli host strain PLR21 for secretion of the gene products.
  • the His-tag purification kit may be used to isolate and purify the recombinant proteins (Novagen). Large scale recombinant His-tag proteins may be purified by affinity chromatography with Hi-trap nickel columns (Pharmacia, Piscataway, NJ) using an automated fast performance liquid chromatography system (Pharmacia, Piscataway, NJ) and recommended manufacturer's procedures for the later generation of polyclonal sera against recovered antigens.
  • the antigens may be cloned into the pIVEX vector system for cell free expression of the antigens in the Rapid Translation System (RTS500; Roche, Indianapolis, IN).
  • RTS500 Rapid Translation System
  • Recombinant proteins may be submitted to a fee for service rabbit polyclonal service to generate polyclonal sera against the recombinant proteins using methods well-known in the art.
  • These antisera may be further tested by Western blot analysis for confirmation of specificity to the recombinant antigen in comparison with whole cell lysates and CFP from M. tuberculosis as described previously (Dobos et al, 2000a and King et al., 2002).
  • DNP dinitrophenyl
  • the DNP molecule may be used like biotin or digoxigenin to label both DNA and proteins for localization of intracellular antigens and has been successfully used to locate DNA regions of HIV-infected cells and to probe for DNP-labeled IgG antibody to detect rare intracellular antigens (Spadoro et al., 1990; Gee et al., 1990). Other methods of delivering the labeled antibody into the infected epithelial cells and macrophages may be used.
  • BioPorter protein delivery system may be used to package the DNP- labeled antibodies according to the manufactures directions.
  • This lipid-based delivery system effectively packages the antibodies through non-covalent interactions, and then may be used to translocate active antibodies into the cytosol of viable eukaryotic cells (without degradation) where then these antibodies dissociate from the carrier molecules and are free to interact with intracellular organelles, proteins, and membranes (Zelphati et al., 2001).
  • This protein delivery system has been successfully used to deliver active proteins into viable A549 epithelial cells (Das et al., 2001; Alcorn et al., 2001; Wu et al., 2001) and PBMCs (Schleef et al., 2001).
  • Infected and uninfected epithelial cells and macrophages may be obtained as described above from cover slips of monolayers (in 24-well tissue culture plates) at early and late stages of the infections (at time points preceding and after significant LDH release and necrosis).
  • Pulse/chase experiments with viable pre/post infected and control cells may be exposed to various concentrations of packaged antibodies against each identified antigen (e.g.
  • Coverslips may be removed from tissue culture wells, washed three times with PBS, and fixed for 20 min with 4% paraformaldehyde in PBS. Coverslips may then be washed tliree times with PBS and permeabilized for 20 min with 0.1% saponin in PBS, and then blocked with 1%) saponin-2.0% bovine serum albumin in PBS (SBP) for 20 min.
  • SBP bovine serum albumin in PBS
  • Anti-dinitrophenyl (DNP) antibodies either anti-rabbit for polyclonal antibody detection or anti-mouse for Mab detection conjugated with Alexa Fluor 546 maleimide (bright orange fluorescence; excitation emission ⁇ 558/573 nm, Molecular Probes inc.) may be added at appropriate dilutions in SBP to the permeabilized cells and allowed to incubate overnight at 4°C. Coverslips may then be washed three times in PBS and blocked again with SBP for 20 min. After washing the blocking reagent off the coverslips, each coverslip may be mounted in 50% glycerol in PBS, sealed to slides, and stored at 4°C until confocal visualization.
  • Alexa Fluor 546 maleimide blue orange fluorescence; excitation emission ⁇ 558/573 nm, Molecular Probes inc.
  • Confocal microscopy may then be used to detect the presence and location of each intracellular antigen in relation to GFP- labeled wild type M. tuberculosis as previously described (Griffiths, 1993; Barker et al., 1997; Teitelbaum et al., 1999) with appropriate modifications necessary for the Zeiss confocal system.
  • mice Primary antibodies are readily available to these human macrophage endosomal markers (BD Biosciences) and the methods of these labeling techniques have been described previously (Barker et al., 1997). Alternatively, the mouse macrophage cell line BMA3.1A7 and goat anti-mouse endosomal antibodies, available from the same source (BD Biosciences), may be used in these proposed experiments. c. Characterization of the role of the genes in the induction of cellular activation cascades.
  • bacterial factors involved in virulence may induce host cell death by activation of host cell factors that subsequently lead to disruption of the host cell plasma membrane and primary necrosis (in contrast to secondary necrosis which is defined as non specific membrane disruptions due to pore forming toxins, hemolysins, etc).
  • intraphagosomal pathogens e.g. Salmonella
  • Salmonella induce host cell necrosis through a programmed primary nec ⁇ otic cell death different than apoptosis (in addition to a apoptotic cell death response to infection that is known to occur) via cellular activation cascades directly related to the host inflammatory response
  • This model would be consistent with M. tuberculosis infection and induction of host cell factors that could cause cell death via programmed necrosis as part of the complex mechanisms in the immunopathology of tuberculosis.
  • IL-l ⁇ The proinflammatory cytokine interleukin-1 (IL-l ⁇ ) was expressed by naive human macrophages and dendritic cells when infected with M. tuberculosis (Giacomini et al., 2001), and from neutrophils and peripheral blood monocytes obtained from pulmonary tuberculosis patients (Wang et al., 2001; Aleman et al., 2001). Likewise, peripheral blood monocytes from pulmonary tuberculosis patients also have an upregulation of inducible nitric oxide synthase (iNOS), and this could be inhibited with a competitive inhibitor of iNOS (Wang et al., 2001).
  • iNOS inducible nitric oxide synthase
  • cytotoxicity of human neutrophils and fibroblast was enhanced with the addition of IL-l ⁇ (Takii et al., 2001; Aleman et al., 2001) even though this cytokine normally stimulates fibroblast growth in the absence of M. tuberculosis infection.
  • NO can also be destructive to host plasma membranes in epithelial cells in high concentration when iNOS is activated as demonstrated by the effects of the tracheal cytotoxin (TST) of Bordetella pertussis (Heiss et al., 1993; Heiss et al., 1993; Flak et al., 2000).
  • TST tracheal cytotoxin
  • This small molecular weight peptide (a 921 -Da disaccharide tefrapeptide released from the cell membrane of the bacteria) induces toxic levels of NO through induction of IL- 1, and this cytotoxicity is related to the lung cytopathology through the destruction of ciliated epithelial cells during the disease whooping cough (Heiss et al., 1994; Flak and Goldman, 1996; Flak et al., 2000).
  • IL- 1 may cause necrosis via induction of iNOS and subsequent elevation of levels of NO leading to membrane disruptions and necrosis of epithelial cells.
  • IL-l ⁇ activation of iNOS which leads to intracellular NO toxicity in B. pertussis infections of human epithelial cells, these findings suggest an additional toxic role for the proinflammatory cytokine IL-l ⁇ induced by M. tuberculosis infection.
  • tuberculosis infection from bacterial mediated primary necrosis both of which may be programmed cell death
  • apoptosis is a protective response to kill M. tuberculosis and primary necrosis is a virulence mechanism for killing host cells.
  • Murine macrophages may be used throughout these studies because they produce greater amounts of NO in vitro (Piddington et al., 2001) and wild type M. tuberculosis survives within these macrophages in the presence of high levels of NO.
  • IL-1 levels from murine macrophages may be measured using mouse IL-l ⁇ and IL-l ⁇ BD OptEIA ELISA's (BD Biosciences) according to the manufactures instructions.
  • IL-1 levels in epithelial cells may be measured using Human IL- l ⁇ BD OptEIA ELISA (BD Biosciences).
  • NO levels may be measured from both cell infections using the Griess reagent (Stuelir and Marietta, 1985) compared to a standard curve of NaNO 2 as previously described by Piddington et al., (2001).
  • We will then inhibit NO through iNOS inhibition using the competitive iNOS inhibitor NG-monomethyl-L- arginine, L-NMMA), and measure the NO and IL-1 levels as described above and cellular necrosis as described in Dobos et al., 2000b.
  • Such studies may be perfo ⁇ ned using cells infected with all nee mutants to compare to wild type levels for comparison.
  • Inhibitor studies may be performed by pre-exposing epithelial cells and macrophages to the caspase- 1 inhibitor, acetyl-Tyr-Val-Ala-Asp-choloromethyl ketone (ac- YVAD-smk; effective concentration is 100-200 uM; Calbiochem).
  • Monolayers may be prepared as described above, pretreated with ac-YVAD-smk for one hour as described by Brennan and Cookson, 2000, and then infected with wild type M. tuberculosis..
  • Levels of IL-1, NO, LDH release, and necrosis from epithelial cells infections may be measured over time as described above to determine the requirement of caspase- 1 and the resultant effects of NO to the necrosis phenotype.
  • Selected nee mutants, complemented nee mutants, and non-revertible nee mutant may be tested similarly if the parent strain is found to activate this cascade to determine the primary nee gene product responsible.
  • necrosis related to cellular activation cascades may also be induced by secondary effects of membrane distortions. DNA damage and strand breakage's are associated with necrosis as well as apoptosis. However, because DNA damage associated with necrosis activates ADP-ribose Polymerase (PARP) to consume NAD+, intracellular pools of ATP are depleted leading to necrotic cell death from non-specific membrane leakage of ions (Ha and Snyder, 1999).
  • PARP ADP-ribose Polymerase
  • necrosis may be measured by simply treating the infected cells with Glycine (Dong et al., 1997) and determining LDH release and necrosis from infected and uninfected epithelial cells and macrophages.
  • Glycine (5mM; Fisher Chemicals) may be used to stop lethal ion fluxes related to necrosis during infection of epithelial cells and macrophages by wild type M. tuberculosis using the methods described by Brennan and Cookson, (2000).
  • Each of these experiments may be performed in the presence and absence of Glycine, and LDH release and necrosis measured as previously described (Dobos et al., 2000b; Appendix).
  • tuberculosis mediated necrosis determine whether this phenotype is principally caused by factors that cause disruption or pore formation of the plasma membrane (suggested by the release of LDH) or by induction of cellular activation cascades (where LDH release may be a secondary phenotype or consequence of cellular activation cascades due to ATP depletion).
  • LDH release may be a secondary phenotype or consequence of cellular activation cascades due to ATP depletion.
  • L. pneumophila based on a similar expression of a contact-dependent cytolytic activity of erythrocytes in vitro (Kirby et al., 1998).
  • the DNA region in L. pneumophila encoding the dot genes was shown to be required for the insertion of pores by L. pneumophila after infection of human macrophages leading to egress from the cells.
  • tuberculosis infected epithelial cells (Dobos et al., 2000b), it is possible that other nee gene products may directly interact with the plasma membranes of host cells as an additional mechanism for necrosis and/or egress from host cells.
  • wild type Erdman may be tested for membrane depolarization in epithelial cells using osmoprotection and dye exclusion assays previously described (Kirby et al., 1998, Krause et al., 1998).
  • Viable bacilli are preferably used because the nee gene products may be expressed only during infection and/or intracellular growth (as may be the case for NecB) or activated upon intracellular secretion within the environment of the phagosome or after escape into the cytosol (as may be the case for Nec-4/Rv3875 and or NecB gene products).
  • Osmoprotection studies may be performed using polyethylene glycol (PEG) to determine the approximate pore size created by M. tuberculosis during infection of epithelial cells and macrophages.
  • PEG polyethylene glycol
  • This method has been used to determine pore size in eukaryotic cell membranes by the differential cytoprotective abilities of different sizes of PEG (Kirby et al., 1998).
  • Monolayers of cells may be preincubated with sized PEG's (PEG1000 to PEG8000; Sigma) and exposed to various MOI's of bacilli (beginning with MOI's of 100 and 500 to reduce the necessary time point for permeation) and incubated at 37°C/5%) CO 2 ..
  • Monolayers may be stained with ethidium and acridine orange at time points predetermined from wild type experiments described above, and visualized for exclusion of the ethidium using a fluorescence microscope with a FITC or rhodamine bandpass filter (Zeiss). Ethidium is excluded from cells with an intact cell membrane whereas acridine orange stains all cells, giving a prediction of the estimated pore size from the cytoprotective PEG's. To characterize the membrane permeation activity further, sized dye exclusion assays may be used to determine the presence of pore-forming molecules according to the methods described by Kirby et al., (1998) and others ( ⁇ oronha et al., 1996).
  • the ability of various-sized membrane-impermeant dyes to gain access to the cytoplasm of osmotically protected epithelial cells or macrophages after infection with wild type M. tuberculosis may be determined using fluorescent microscopy as described for the dye exclusion assay.
  • the dye exclusion assays are not useful, we will use the LDH release assay after pre-exposure of cells with the cytoprotective PEG's at various time points as previously described.
  • dye-exclusion experiments for hemoglobin release from sheep erythrocytes may also be preformed by measuring differential hemoglobin release as described previously (King et al., 1993; Kirby et al., 1998).
  • Texas Red-tagged dextrans 3, 40, 70, and 2,000 kDa
  • Texas Red-tagged ovalbumin each available from Molecular Probes inc.
  • individual Texas Red-tagged molecules may be packaged with the BioPorter and/or Chariot protein delivery agents as described above, exposed to GFP-labeled M.
  • tuberculosis wild type and selected nee mutant infected and uninfected cells incubated with monolayers on cover slips, and visualized for co-localization with M. tuberculosis phagosomes as described above and by Teitelbaum et al., 1999b).
  • EXAMPLE 8 Exhaustive screening of the M. tuberculosis transposon library for mutants defective in epithelial cell permeation and necrosis.
  • nee genes may be identified using the same mutant screen and analyses described above. Legionella pneumophila has over 23 dot/icm genes, the majority of which are involved in the cell membrane permeation, the cytotoxic response and egress from the host cell. Likewise, Salmonella spp. expresses several genes as part of a type III secretion apparatus involved in host cell invasion, membrane trafficking, and cell death via necrosis. It is plausible the pathogen M. tuberculosis has at least as many nee related genes, and many of these genes may be involved in related pheno types.
  • necA, Rv3875, or necB genes may be involved in transport of another pore forming or membrane distorting nee gene product or each other (e.g. the necA gene and the Rv3875 may be required together to induce host cell membrane distortions) rather than directly involved in the membrane disruption of the infected cells.
  • the recombinant library of M. tuberculosis Erdman may be further screened and analyzed for selection of additional mutants with defects in membrane permeation and necrosis of epithelial cells relevant to virulence in the SCID model as described above.
  • nee mutants that appear virulent in the intravenous infection of SCID mice may be tested by the aerosol infection SCID mouse model to determine if these mutants warrant further study using the methods described above.
  • These additional nee genes may be involved in the regulation, secretion and/or modification of nee gene products and thus are important to dedicate additional resources and time to perform.
  • a hygromycin- tagged Mariner transposon phage library may be screened for mutants defective in necrosis as described above.
  • Identifying the transposon disrupted gene, generating complemented and/or non-revertible mutants in each gene, and testing each as described above for expression during infection and the interaction of nee gene products may be carried out as described above.
  • Selected mutants and complemented clones may be studied further in both the in vitro and in vivo models described above.
  • Each mutant pair and nee gene product may be studied based on a selection criteria of which nee genes are directly involved in virulence and pathology in the SCID and Balb/c mouse models. Only those selected nee genes that are observed to be critical to virulence in the SCID mouse model will first be chosen for additional study in the mouse models as described above for the analysis of necA and necB.
  • M. tuberculosis Erdman strain
  • Middlebrook 7H9 medium with OADC supplement Remel, Lenexa, KS
  • OADC supplement Remel, Lenexa, KS
  • Whole cells were lysed in phenol using a cell disrupter (Mini-Beadbeater, type BX-4; Biospec Products, Bartlesville, OK).
  • the pellet was resuspended in 10 mM Tris buffer, pH 8.0, 1 mM EDTA and genomic DNA obtained from cell lysates using phenol-chloroform extraction, followed by ethanol precipitation (WHO, 2001; Portaels et al. Ed.)
  • Cloning, expression and purification of recombinant Rv3351c antigen
  • Rv3351c gene was PCR amplified from M. tuberculosis (Erdman strain) gDNA using the following pairs of oligonucleotide primers (the portions of each primer in lowercase contain restriction endonuclease sites, EcoRV on the forward primer and Xlwl on the reverse primer, for directional insertion of the fragments into the expression vector): 5'- att gac gat ate ATG CTG GCG AGC TGC CCG GCG-3' and 5'-ttc aga etc gag CCG CCG CGG CGT GCG CCG AAC-3' (using an initial hot start of 95°C for 5 min, followed by 25 cycles of 95°C for 1 min, 66°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 7 min).
  • Amplified products were gel purified and ligated in frame to the T7 promoter and His-tag component of the pET29(a)+ vector (Novagen, Inc., Madison, WI).
  • the expression construct was transformed into NovaBlue E. coli host cells (Novagen) for propagation and storage.
  • Miniprep DNA was prepared for restriction analysis to check for the presence of inserts, and for DNA sequencing, to confirm the in-frame position of the cloned insert to the T7 promoter and His-tag sequence.
  • Confirmed expression constructs were transformed into BL21 (DE3) E. coli host cells (Novagen) for expression and secretion of the gene products.
  • the expressed recombinant protein was secreted in inclusion bodies, thus purified using the batch purification method under denaturing conditions, as contained in the Ni- NTA His.Bind® resins purification protocol (Novagen). Recombinant protein was stained with gelcode 6xHis protein tag stain (Pierce Biotechnology Inc., Rockford, IL), to confirm the presence of His tagged protein.
  • gelcode 6xHis protein tag stain Pieris Biotechnology Inc., Rockford, IL
  • Recombinant proteins were resolved using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (Laemmli, 1970) with 10 - 20%) gradient 10-well pre-cast gels (Novex, San Diego, CA). Western blot analysis was carried out using standard protocols as described previously (Dobos, et al, 2000). SDS-PAGE fractionated recombinant Rv3351c proteins were transferred onto 0.45 ⁇ m nitrocellulose membranes (Schleicher & Schuell Inc., Keene, N.H.) and blocked overnight at 4°C with 1% blocking reagent (Roche Molecular Biochemicals, Indianapolis, IN).
  • SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
  • Nitrocellulose sheets of transfe ⁇ ed recombinant proteins were cut into strips for use in antibody detection.
  • TB patient serum antibody to recombinant Rv3351c products were analyzed by probing nitrocellulose strips with serum samples at 1:50 dilution.
  • Non- tuberculosis patient sera obtained from the United States, which served as controls were tested at the same dilution.
  • Bound antibody was detected with alkaline phosphatase conjugated goat anti-human IgG (H+L) antibody (Pierce Biotechnology Inc., Rockford, IL) and substrates nitro blue tefrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl-phospate, toluidine salt; BCIP; Roche Molecular Biochemicals, Indianapolis, IN), according to manufacturer's instruction. All serum samples were tested in triplicate for reproducibility and confirmation of the specific antibody reactivity to M. tuberculosis Rv3351c recombinant protein. e. Statistical Analysis:
  • the protein was purified using Ni NTA His bind resins, and it migrated at a MW of 30 kDa on a 10 - 20% gradient 10-well pre-cast gel (Novex, San Diego, CA).
  • the resulting purified recombinant protein was partially pure since two major contaminating E. coli proteins were still visible on Coomassie stain. Since serum antibody cross-reactivity to bacterial proteins is common in TB endemic areas, the identity of the expressed recombinant protein used in this study was rigorously confirmed.
  • the expression construct was sequenced and the DNA sequence analysis confi ⁇ ned the presence of a high fidelity Rv3351c gene.
  • the recombinant 6xHis tagged fusion protein was positively identified via gelcode 6xHis tag staining ( Figure 15), indicative that it was properly expressed along with the 6xHis tag. Furthermore, the amino acid spectra of the recombinant Rv3351c protein was generated, analyzed and blasted against the protein data base, and it confirmed that the amino acid composition is specific to Rv3351c protein of Mycobacterium tuberculosis
  • Recombinant protein from the Rv3351c gene expressed in Escherichia coli was used to screen the sera of M. tuberculosis infected patients by Western blot ( Figure 16).
  • Preliminary analysis using pooled sera of twenty confirmed TB patients from an old stock of WHO sera bank recognized the presence of the purified Rv3351c recombinant protein.
  • the present invention provides in part novel necrosis-deficient mutants of M. tuberculosis bacteria, as well as novel M. tuberculosis antigens. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appendant claims are not intended to claim all such embodiments and variations, and the full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

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Abstract

La présente invention concerne de nouveaux mutants de bactéries de la tuberculose dépourvus d'activité nécrosante ainsi que des antigènes identifiés avec ceux-ci. Cette invention concerne aussi des techniques d'utilisation de ces mutants et de ces antigènes dans le diagnostic, le traitement et la prévention de l'infection tuberculeuse.
PCT/US2004/002056 2003-01-24 2004-01-26 Mutants de bacteries de la tuberculose depourvus d'activite necrosante Ceased WO2004067718A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1846024A4 (fr) * 2005-01-12 2009-07-29 Einstein Coll Med Mutants mycobacteriens afectant l'apoptose chez l'hôte
EP2535426A3 (fr) * 2006-03-02 2013-07-24 The Uab Research Foundation Détection, traitement de maladies mycobactériennes et découverte de médicaments
JP2015199771A (ja) * 2009-04-24 2015-11-12 スタテンス セールム インスティトゥート 再活性化を予防する結核ｔｂワクチン

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CA2453173C (fr) * 2001-07-04 2013-12-10 Health Protection Agency Antigenes mycobacteriens exprimes pendant la latence

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1846024A4 (fr) * 2005-01-12 2009-07-29 Einstein Coll Med Mutants mycobacteriens afectant l'apoptose chez l'hôte
AU2006204907B2 (en) * 2005-01-12 2011-08-11 Albert Einstein College Of Medicine, Inc. Mycobacterial mutants affecting host apoptosis
US8394388B2 (en) 2005-01-12 2013-03-12 Albert Einstein College Of Medicine Of Yeshiva University Mycobacterial mutants affecting host apoptosis
EP2535426A3 (fr) * 2006-03-02 2013-07-24 The Uab Research Foundation Détection, traitement de maladies mycobactériennes et découverte de médicaments
US8603751B2 (en) 2006-03-02 2013-12-10 The Uab Research Foundation Mycobacterial disease detection, treatment, and drug discovery
JP2015199771A (ja) * 2009-04-24 2015-11-12 スタテンス セールム インスティトゥート 再活性化を予防する結核ｔｂワクチン
US10519202B2 (en) 2009-04-24 2019-12-31 Statens Serum Institut Tuberculosis TB vaccine to prevent reactivation

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