CA2170148A1 - Dna molecule encoding for cellular uptake of mycobacterium tuberculosis - Google Patents

Abstract

The present invention relates to a DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages. The protein encoded by this gene fragment is useful in vaccines to prevent infection by Mycobacterium tuberculosis, while the antibodies raised against this protein can be employed in passively immunizing those already infected by the organism. Both these proteins and antibodies may be utilized in diagnostic assays to detect Mycobacterium tuberculosis in tissue or bodily fluids. The protein of the present invention can be associated with various other therapeutic materials, for administration to mammals, particularly humans, to achieve uptake of those materials by such cells.

Description

W095/06726 _ 1 _ PCT~S~ 9 DNA MOLECULE ENCODING FOR CELLULAR UPTAKE OF
MYCOBACTERIUM TUBERCULOSIS
.

FIELD OF THE lNV~N-llON

The present invention relates to a DNA molecule encoding for uptake of Mycobacterium tuberculosis and its use in drugs, vaccines, and diagnostic tests.

R~CR~ROUND OF THE lNV~NLlON

Tuberculosis is the leading cause of death in the world with an estimated 9 million new cases of tuberculosis and 2.9 million deaths occurring from the disease each year.
In the United States, the steadily declining incidents of tuberculosis has been reversed since 1985. This problem is compounded by the increasing incidence of drug-resistant strains of Mycobacterium tuberculosis.
Recent outbreaks of tuberculosis have involved settings in which a large number of HIV-infected persons resided in close proximity (e.g., AIDS wards in hospitals, correctional facilities, and hospices). Transmission of tuberculosis to health care workers occurred in these outbreaks; 18 to 50~ of such workers showed a conversion in their skin tests. See F. Laraque et. al., "Tuberculosis in HIV-Infected Patients," The AIDS Reader (September/October 1992), which is hereby incorporated by reference.
There are two basic clinical patterns that follow infection with Mycobacterium tuberculosis.
In the majority of cases, inhaled tubercle bacilli ingested by phagocytic alveolar macrophages are either directly killed or grow intracellularly to a limited extent in local lesions called tubercles. Infrequently in children and immunocompromised individuals, there is early hematogenous dissemination with the formation of small W095/06726 2 ¦~ O 1 4 8 PCT~S94/09863 miliary (millet-like) lesions or life-threatening meningitis. More commonly, within 2 to 6 weeks after infection, cell-mediated immunity develops, and infiltration into the lesion of immune lymphocytes and activated macrophages results in the killing of most bacilli and the walling-off of this primary infection, often without symptoms being noted by the infected individual. Skin-test reactivity to a purified protein derivative ("PPD") of tuberculin and, in some cases, X-ray evidence of a healed, calcified lesion provide the only evidence of the infection.
Nevertheless, to an unknown extent, dormant but viable Mycobacterium tuberculosis bacilli persist.
The second pattern is the progression or breakdown of infection to active disease. Individuals infected with Mycobacterium tuberculosis have a 10~ lifetime risk of developing the disease. In either case, the bacilli spread from the site of initial infection in the lung through the lymphatics or blood to other parts of the body, the apex of the lung and the regional lymph node being favored sites.
Extrapulmonary tuberculosis of the pleura, lymphatics, bone, genito-urinary system, meninges, peritoneum, or skin occurs in about 15~ of tuberculosis patients. Although many bacilli are killed, a large proportion of infiltrating phagocytes and lung parenchymal cells die as well, producing characteristic solid caseous (cheese-like) necrosis in which bacilli may survive but not flourish. If a protective immune response dominates, the lesion may be arrested, albeit with some residual damage to the lung or other tissue. If the necrotic reaction expands, breaking into a bronchus, a cavity is produced in the lung, allowing large numbers of bacilli to spread with coughing to the outside.
In the worst case, the solid necrosis, perhaps a result of released hydrolases from inflammatory cells, may liquefy, which creates a rich medium for the proliferation of bacilli, perhaps reaching 109 per milliliter. The W095/06726 21 701 q 8 PCT~S9~ 5~63 pathologic and inflammatory processes produce the characteristic weakness, fever, chest pain, cough, and, when a blood vessel is eroded, bloody sputum.
Ignorance of the molecular basis of virulence and pathogenesis is great. It has been suggested that the establishment of molecular evidence regarding avirulent strains, the identification and cloning of putative virulence genes of the pathogen, and the demonstration that virulence can be conveyed to an avirulent strain by those genes is necessary. Although avirulent strains of Mycobacterium tuberculosis exist, the nature of the mutations is unknown. Not a single gene involved in the pathogenesis of tuberculosis has been defined in the prior art. The molecular bases of invasion of host cells, intracellular survival, growth, spread, or tissue tropism also have not been known. None of the targets of existing drugs has been characterized at a molecular level, and the mechanism of resistance to any drug has not been defined; no new mycobacterial target for drug development has been characterized in 20 years.
There have been many prescribed treatment regimens for tuberculosis. The regimen recommended by the U.S.
Public Health Service and the American Thoracic Society is a combination of isoniazid, rifampicin, and pyrazinamide for two months followed by administration of isoniazid and rifampicin for an additional four months. In persons with HIV infection, isoniazid and rifampicin treatment are continued for an additional seven months. This treatment, called the short-course chemotherapy, produces a cure rate of over 90~ for patients who complete it. Treatment for multi-drug resistant tuberculosis requires addition of ethambutol and/or streptomycin in the initial regimen, or second line drugs, such as kanamycin, amikacin, capreomycin, ethionamide, cyclcoserine, PAS, and clofazimin. New drugs, such as ciprofloxacin and ofloxacin can also be used. For WO95/06726 ~4~ rcT~sg1~09~53 individuals infected with conventional Mycobacterium tuberculosis and showing PPD positive results, chemoprophylaxis with isoniazid has been about 90~ effective in preventing the disease. Tuberculosis and these treatments are discussed in more detail in B. Bloom et. al., "Tuberculosis: Commentary on a Reemergent Killer," Science, 257:1055-64 (1992); "Control of Tuberculosis in the United States," American Thoracic Society, 146:1623-33 (1992); Citv Health Information, vol. 11 (1992), which is hereby incorporated by reference.
Although the currently used treatments for tuberculosis have a relatively high level of success, the need remains to improve the success rate for treating this disease. Moreover, in view of the ever-increasing level of Mycobacterium tuberculosis strains which are resistant to conventional treatment regimens, new types of treatment must be developed. In high tuberculosis endemic areas, both in the United States and abroad, such resistant strains are becoming increasingly present.
SUMMARY OF THE lNv~L.llON

The present invention relates to an isolated DNA
molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages as well as an isolated protein or polypeptide encoded by that isolated DNA molecule. The molecule can be inserted as a heterologous DNA in an expression vector forming a recombinant DNA expression system for producing the protein or peptide. Likewise, the heterologous DNA, usually inserted in an expression vector to form a recombinant DNA
expression system can be incorporated in a cell to achieve this objective.
The isolated protein or polypeptide of the present invention can be combined with a pharmaceutically-acceptable WO95/06726 Z~ 7~ l~o PCl~S9~/~5i63 carrier to form a vaccine or used alone for administration to m~mm~l S, particularly humans, for preventing infection by Mycobacterium tuberculosis. Alternatively, the protein or polypeptide of the present invention can be used to raise an antibody or a binding portion thereof. The antibody or binding portion thereof may be used alone or combined with a pharmaceutically-acceptable carrier to treat m~mm~l S, particularly humans, already exposed to Mycobacterium tuberculosis to induce a passive immunity to prevent disease occurrence.
The protein or polypeptide of the present invention or the antibodies or binding portions thereof raised against them can also be utilized in a method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids. When the protein or polypeptide are utilized, they are provided as an antigen. Any reaction with the antigen or the antibody is detected using an assay system which indicates the presence of Mycobacterium tuberculosis in the sample. Alternatively, Mycobacterium tuberculosis can be detected in such a sample by providing a nucleotide sequence of the gene conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages or a fragment thereof as a probe in a nucleic acid hybridization assay or a gene amplication detection procedure (e.g., using a polymerase chain reaction procedure). Any reaction with the probe is detected so that the presence of Mycobacterium tuberculosis in the sample is indicated.
The protein or polypeptide of the present invention can also be used for purposes unrelated to the treatment or detection of Mycobacterium tuberculosis. More particularly, the ability of that protein or polypeptide to confer on Mycobacterium tuberculosis an ability to enter m~m~l ian cells can be utilized to permit such cells to uptake other materials. This can be achieved with a product W095/06726 ~ 0~ 4~ PCT~S94/09863 ~ - 6 -that includes a material for uptake by m~mm~l ian cells and the protein or polypeptide of the present invention associated with that material.
Isolation of the DNA molecule of the present s invention constitutes a significant advance in the treatment and detection of such bacteria. It also provides the basis for a vaccine to prevent infection by Mycobacterium tuberculosis and a pharmaceutical agent for passive immunization for those exposed to Mycobacterium tuberculosis. The protein utilized in the vaccine or to produce the pharmaceutical agent can be produced at high levels using recombinant DNA technology.
In diagnostic applications, the protein or polypeptide of the present invention as well as antibodies and binding portions thereof against them permit rapid determination of whether a particular individual is infected with Mycobacterium tuberculosis. Moreover, such detection can be carried out without requiring an examination of the individual being tested for an antibody response.
Aside from the development of treatments and diagnostic tools for Mycobacterium tuberculosis, the present invention's ability to confer entry of such organisms into m~mm~l ian cells has significant utility in therapeutic treatments requiring the introduction of materials into cells, particularly to macrophages. By associating the protein or polypeptide of the present invention with pharmaceutical agents, such agents can be rapidly introduced into cells for treatment thereof. The enhanced cellular uptake of such products can reduce drug dosages, thus reducing toxicity and cost. For example, in conventional cancer treatment, drug toxicity is a major problem due to the requirement for administration of large dosages; the present invention has the potential to reduce such high dosage levels while enabling delivery of equivalent or higher drug levels intracellularly.

W095~6726 ~1 7l ~8 PCT~S~ 9~3 Furthermore, binding the protein or polypeptide of the present invention to DNA fragments can be utilized in conjunction with gene therapy regimens. In particular, the ability of the encoded product of the DNA molecule of the 5 present invention to augment uptake into macrophages provides an opportunity to deliver genes specifically to macrophages. Such a system can be used to induce not only humoral immunity but cell-mediated immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures lA, lB, and lC are thin-section electron micrographs of HeLa cells infected with Mycobacterium tuberculosis strain, including H37Ra (ATCC25177) (Figure lA), and the invasive recombinant strain E. coli XL1-Blue (pZX7) (Figure lB and lC). An electron-transparent zone surrounds the Mycobacterium tuberculosis organism (arrow in Figure lA). The cells were incubated with Mycobacterium tuberculosis strain for 72 hours in Figure lA
and with XL1-Blue (pZX7) for 7.5 hours in Figures lB and lC.
Multiple organisms are visible in Figure lC, suggesting bacterial proliferation inside phagosomes. The bars represent 0.5 ~m.
Figure 2 shows the construction of unidirectional deletional subclones (pZX7.3, pZX7.4, pZX7.5, and pZX7.6) and Bam HI-Pst I (pZX7.1), Pst I-HinD III (pZX7.2), and Bam HI-Eco RI (pZX7.7) subclones from the original vector pZX7.
The black bars represent the Mycobacterium tuberculosis DNA
sequences, and the white bars represent pBluescript sequences. The subclone vectors were transferred into E.
coli XL1-Blue and then incubated with these transformed strains for 6 hours with a HeLa cell monolayer.
Figures 3A, 3B, and 3C are thin-section electron micrographs of human macrophages exposed to the invasive recombinant E. coli clone XL1-Blue(pZX7) for 3 hours W095/06726 ~ ~ 0 ~ 4~ PCT~S94/09863 (Figure 3A) and 24 hours (Figure 3B) compared with cells exposed to nonpathogenic E. coli XLl-Blue(pBluescript) for 24 hours (Figure 3C). The bacteria become compartmentalized, surrounded by layers of membrane inside the macrophage (Figure 3B). No bacteria were visible after 24 hours by electron microscopy in macrophages exposed to XL1-Blue(pBluescript). The bars represent 1 ~m.
Figure 4 shows the SDS-polyacrylamide gel electrophoresis of an acetone-precipitated soluble fraction of bacterial cell sonicate. The polypeptides were analyzed in a 9~ gel (left): molecular size standards (lane 1), E.
coli XLl-Blue with a vector (pZN7) containing an unrelated Mycobacterium tuberculosis DNA fragment between the Bam HI-Eco RI pBluescript cloning sites (lane 2), and XLl-Blue(pZX7) (lane 3). Analysis in an 8~ gel (right): XL1-Blue containing a vector (pZX7.8) with a two-base frameshift introduced 12 bases upstream from the Bam HI cloning site in pZX7 (lane 1) and XL1-Blue(pZX7) (lane 2). Molecular sizes are indicated at the far right. We detected a 52-kD
polypeptide in the soluble protein fraction of XL1-Blue(pZX7) (arrow). A protein of about 50 kD is expressed by XL1-Blue containing pZX7.8. The expression of the 52-kD
protein was always associated with HeLa cell interaction of the recom~binant E. coli clone.
DETATTTm DESCRIPTION OF THE lNv~ lON

The present invention relates to an isolated DNA
molecule conferring on Mycobacterium tuberculosis an ability to enter m~mm~lian cells and to survive within macrophages.
This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows:

W095/06726 17.¦ q8 PCT~S91~!~9563 GGATCGAATT GCTGGCCTTT GGCGGGCGAT TCGTGGAGAT CGCCCGTAGA AAGGTTCGCG

GACGCCAAGG CCGCCGCAGA CCGCCATAAA CGTAGTTGAC CAGGTGGTCT TGACTGGGGC

CGGACACCGA CGTGAACGAG GCGACCCGAT CCGCGTTACA TCCACCTGAT TCCGGCAAAT

GTGAACGCCG ACATCAAGGC GACCACGGTG TTCGGCGGTA AGTATGTGTC GTTGACCACG

CCGAAAAACC CGACAAAGAG GCGGATAACG CCAAAAGACG TCATCGACGT ACGGTCGGTG

ACCACCGAGA TCAACACGTT GTTCCAGACG CTCACCTCGA TCGCCGAGAA GGTGGATCCG

GTCAAGCTGA ACCTGACCCT GAGCGCGGCC GCGGAGGCGT TGACCGGGCT GGGCGATAAG

TTCGGCGAGT CGATCGTCAA CGCCAACACC GTTCTGGATG ACCTCAATTC GCGGATGCCG

CAGTCGCGCC ACGACATTCA GCAATTGGCG GCTCTGGGCG ACGTCTACGC CGACGCGGCG

CCGGACCTGT TCGACTTTCT CGACAGTTCG GTGACCACCG CCCGCACCAT CAATGCCCAG

CAAGCGGAAC TGGATTCGGC GCTGTTGGCG GCGGCCGGGT TCGGCAACAC CACAGCCGAT

GTCTTCGACC GCGGCGGGCC GTATCTGCAG CGGGGGGTCG CCGACCTGGT CCCCACCGCC

W095/067~6 ~ PCT~S94/09863 ACCCTGCTCG ACACTTATAG CCCGGAACTG TTCTGCACGA TCCGCAACTT CTACGATGCC

GATCGACCTG ACCGCGGGGC TGCCGCATAG GCCCGGAGTG GTTCGCGATC GGCGAGGCGC

ACGTCAAAGT GATTCGCGCC ~lllllCGCC CACCTGCCCG CCGCGGTGGA TGTGTCCACC

CGCCAGGCCG CCGAAGCCGA CCTGGCCGGC AAAGCCGCTC AATATCGTCC CGACGAGCTG

GCCCGCTACG CCCAGCGGGT CATGGACTGG CTACACCCCG ACGGCGACCT CACCGACACC

GAACGCGCCC GCAAACGCGG CATCACCCTG AGCAACCAGC AATACGACGG CATGTCACGG

CTAAGTGGCT ACCTGACCCC CCAAGCGCGG GCCACCTTTG AAGCCGTGCT AGCCAAACTG

GCCGCCCCCG GCGCGACCAA CCCCGACGAC CACACCCCGG TCATCGACAC CACCCCCGAT

GCGGCCGCCA TCGACCGCGA CACCCGCAGC CAAGCCCAAC GCAACCACGA CGGGCTGCTG

GCCGGGCTGC GCGCGCTGAT CCGTCATCCT GCCATCTCGG CCCTCGGCGC CGCCAACTCC

AGGTGCTGTG CGGTCCACGC CGAACGCATG CACGCGATCT CGAATTGGTT GGCACCGTAT

TCGGGATGGA ACTGCTCGAT AGCGATGCCT GCTGCCGTTG CCGCGGCGTT GACATCGCGG

WO95t06726 'l~q~ PCT~S94/09863 ACGAACGCCT CGTGCTCGAG CACCCCGGCG ACACCGTACT GCGCCCACAG CGTCGAAGGC

AGCCGCTGGC CGTCCGCGTC GACCAAGAGG AATTC

The DNA molecule encodes for a polypeptide having a molecular weight of about 50 to 55 kilodaltons, preferably 52 kilodaltons. The amino acid sequence, deduced from the nucleotide sequence corresponding to SEQ. ID. No. 1, represents a highly hydrophilic protein with a hydrophobic region at its carboxy terminus. It could be a secreted protein, a cytoplasmic protein, or a surface protein with its carboxy terminus attached to the outer membrane of the organism. It is believed that this protein or polypeptide has the deduced amino acid sequence corresponding to SEQ.
ID. No. 2 as follows:

Gly Ser Asn Cys Trp Pro Leu Ala Gly Asp Ser Trp Arg Ser Pro Val Glu Arg Phe Ala Asp Ala Lys Ala Ala Ala Asp Arg His Lys Arg Ser Xaa Pro Gly Gly Leu Asp Trp Gly Arg Thr Pro Thr Xaa Thr Arg Arg Pro Asp Pro Arg Tyr Ile His Leu Ile Pro Ala Asn Val Asn Ala Asp Ile Lys Ala Thr Thr Val Phe Gly Gly Lys Tyr Val Ser Leu Thr Thr Pro Lys Asn Pro Thr Lys Arg Arg Ile Thr Pro Lys Asp Val Ile Asp W095/06726 ~ ~8 ~ PCT~S~ Oj~63 Val Arg Ser Val Thr Thr Glu Ile Asn Thr Leu Phe Gln Thr Leu Thr er Ile Ala Glu Lys Val Asp Pro Val Lys Leu Asn Leu Thr Leu Ser Ala Ala Ala Glu Ala Leu Thr Gly Leu Gly Asp Lys Phe Gly Glu Ser Ile Val Asn Ala Asn Thr Val Leu Asp Asp Leu Asn Ser Arg Met Pro 145 . 150 155 160 ln Ser Arg His Asp Ile Gln Gln Leu Ala Ala Leu Gly Asp Val Tyr la Asp Ala Ala Pro Asp Leu Phe Asp Phe Leu Asp Ser Ser Val Thr hr Ala Arg Thr Ile Asn Ala Gln Gln Ala Glu Leu Asp Ser Ala Leu Leu Ala Ala Ala Gly Phe Gly Asn Thr Thr Ala Asp Val Phe Asp Arg Gly Gly Pro Tyr Leu Gln Arg Gly Val Ala Asp Leu Val Pro Thr Ala hr Leu Leu Asp Thr Tyr Ser Pro Glu Leu Phe Cys Thr Ile Arg Asn he Tyr Asp Ala Asp Arg Pro Asp Arg Gly Ala Ala Ala Xaa Ala Arg er Gly Ser Arg Ser Ala Arg Arg Thr Ser Lys Xaa Phe Ala Pro Phe W095/06726 1 7l q~ PCr/US94/09863 Phe Ala HiS Leu Pro Ala Ala Val Asp Val Ser Thr Arg Gln Ala Ala Glu Ala Asp Leu Ala Gly Lys Ala Ala Gln Tyr Arg Pro Asp Glu Leu Ala Arg Tyr Ala Gln Arg Val Met Asp Trp Leu His Pro Asp Gly Asp Leu Thr Asp Thr Glu Arg Ala Arg Lys Arg Gly Ile Thr Leu Ser Asn Gln Gln Tyr Asp Gly Met Ser Arg Leu Ser Gly Tyr Leu Thr Pro Gln Ala Arg Ala Thr Phe Glu Ala Val Leu Ala Lys Leu Ala Ala Pro Gly Ala Thr Asn Pro Asp Asp His Thr Pro Val Ile Asp Thr Thr Pro Asp Ala Ala Ala Ile Asp Arg Asp Thr Arg Ser Gln Ala Gln Arg Asn His Asp Gly Leu Leu Ala Gly Leu Arg Ala Leu Ile Arg His Pro Ala Ile Ser Ala Leu Gly Ala Ala Asn Ser Arg Cys Cys Ala Val His Ala Glu Arg Met His Ala Ile Ser Asn Trp Leu Ala Pro Tyr Ser Gly Trp Asn Cys Ser Ile Ala Met Pro Ala Ala Val Ala Ala Ala Leu Thr Ser Arg W095/06726 ~1 ~ 4 - 14 - PCT~S~ .S3 Thr Asn Ala Ser Cys Ser Ser Thr Pro Ala Thr Pro Tyr Cys Ala His Ser Val Glu Gly Ser Arg Trp Pro Ser Ala Ser Thr Lys Arg Asn In the immediately-preceding sequence, Xaa signifies a stop codon. Production of this isolated protein or peptide is preferably carried out using recombinant DNA technology.
The protein or peptide is believed to have one or more antigenic determinants conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.
The protein or polypeptide of the present invention is preferably produced in purified form by conventional techniques. Typically, the protein of the present invention is secreted into the growth medium of recombinant E. coli. To isolate the protein, the E. coli host cell carrying a recomn;n~nt plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernantant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.
The DNA molecule conferring on Mycobacterium tuberculosis an ability to enter m~mm~lian cells and to survive within macrophages can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains W095/06726 ~ ~8 PCT~S94/09863 the necessary elements for the transcription and translation of the inserted protein-coding sequences.
U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtll, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II
SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F.W. Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression TechnoloqY vol. 185 (1990), which is hereby incorporated by reference) and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as - described by Maniatis et al., Molecular Clonina: A
Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference.

W095/06726 2~ t4~ PCT~S91/09-3 A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used.
Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; m~mm~l ian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA
transcription and messenger RNA (mRNA) translation).
Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA
synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (SD) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA
that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3'-end of the 16S rRNA
(ribosomal RNA) and probably promote binding of mRNA to W095~67Z6 21701~8 PCT~S94~9863 ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in EnzYmoloqy, 68:473 (1979), which is hereby incorporated by 5 reference.
Promotors vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promotors may be used. For instance, when cloning in E.
coli, its bacteriophages, or plasmids, promotors such as the T7 phage promoter, lac promotor, trp promotor, recA
15 promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others, including but not limited, to lacW5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments.
Additionally, a hybrid trp-lacW5 ( tac) promotor or other E.
col i promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG
(isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity W095t06726 - o PCTtUS94/09863 ~4'~ -of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecule conferring on Mycobacterium tuberculosis an ability to enter m~mm~l ian cells and to survive within macrophages has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, m~mm~l ian cells, and the like.
Generally, the human immune system responds to infection by pathogenic bacteria by producing antibodies that bind to specific proteins or carbohydrates on the bacterial surface. The antibodies stimulate binding to macrophages which have receptors that bind to the Fc region of the antibodies. Other serum proteins, called complement, coat the foreign particle and stimulate their ingestion by binding to specific surface receptors on the macrophage.
Once the particle is bound to the surface of the macrophage, the sequential process of ingestion begins by continual apposition of a segment of the plasma membrane to the W095/06726 1 7l ~8 PCT~S91/0~6~

particle surface. Surface receptors on the membranes then interact with ligands distributed uniformily over the - particle surface to link the surfaces together. The macrophage enveloping the particle is then delivered to lysosomes where the particle is ingested.
Some organisms are ingested (i.e. undergo uptake) by macrophages but are not killed. Amongst these is Mycobacterium tuberculosis. As a result, such organisms are able to survive indefinitely within macrophages and, when they escape from the macrophage, cause active tuberculosis.
In view of the present invention's determination of the nucleotide sequence conferring on Mycobacterium tuberculosis an ability to enter mAmmAlian cells and to survive within macrophages, the molecular basis for Mycobacterium tuberculosis uptake is suggested. With this information and the above-described recombinant DNA
technology, a wide array of therapeutic and/or prophylatic agents and diagnostic procedures for, respectively, treating and detecting Mycobacterium tuberculosis can be developed.
For example, an effective amount of the protein or polypeptide of the present invention can be administered alone or in combination with a pharmaceutically-acceptable carrier to humans, as a vaccine, for preventing infection by Mycobacterium tuberculosis. Alternatively, it is possible to administer to individuals exposed to Mycobacterium tuberculosis with an effective amount of an antibody or binding portion thereof against that protein or polypeptide as a passive immunization. Such antibodies or binding portions thereof are administered alone or in combination with a pharmaceutically-acceptable carrier to effect short term treatment of individuals who may have been recently exposed to Mycobacterium tuberculosis.
Antibodies suitable for use in inducing passive immunity can be monoclonal or polyclonal.

W095/06726 ~ PCT~S3l/'~3~53 Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest (i.e. the protein or peptide of the present invention) either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vi tro to produce large quantities of antibody. A
description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature 256:495 (1975), which is hereby incorporated by reference.
~mm~l ian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. The virus is carried in appropriate solutions or adjuvants. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (See Milstein and Kohler, Eur. J. Immunol.
6:511 (1976), which is hereby incorporated by reference).
This immortal cell line, which is preferably murine, but may also be derived from cells of other m~mm~l ian species, W095/06726 ~1 7~1 ~8 PCT~S94/09863 including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 ~l per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis.
The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody.
Ultimately, the rabbits are euthenized with pentobarbitol 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.
The vaccines and passive immunization agents of this invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in W095/06726 ~ PCT~S9l~'09.~3 ~ - 22 -solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the protein or peptide of the present invention or the antibody or binding portion thereof of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents such as, cornstarch, potato starch, or alginic acid, and a lubricant like stearic acid or magnesium stearate.
The protein or polypeptide of the present invention or the antibody or binding portion thereof of this invention may also be administered in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
For use as aerosols, the protein or polypeptide of the present invention or the antibody or binding portion thereof of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present W095/06726 7l q8 PCT~S~ 963 invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
In yet another aspect of the present invention, the protein or polypeptide of the present invention can be used as antigens in diagnostic assays for the detection of Mycobacterium tuberculosis body fluids. Alternatively, the detection of that bacillus can be achieved with a diagnostic assay employing antibodies or binding portions thereof raised by such antigens. Such techniques permit detection of Mycobacterium tuberculosis in a sample of the following tissue or body fluids: blood, spinal fluid, sputum, pleural fluids, urine, bronchial alveolor lavage, lymph nodes, bone marrow, or other biopsied materials.
In one embodiment, the assay system has a sandwich or competitive format. Examples of suitable assays include an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion pricipitan reaction assay, an immunodiffusion assay, an agglutination assay, a florescent immunoassay, a protein A immunoassay, or an immunoelectrophoresis assay.
In an alternative diagnostic embodiment of the present invention, the nucleotide sequence of the isolated DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages may be used as a probe in nucleic acid hybridization assays for the detection of Mycobacterium tuberculosis in various patient body fluids. The nucleotide sequence of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, J. Mol. Biol., 98:508 (1975)); Northern blots (Thomas et al., Proc. Nat'l Acad. Sci. USA, 77:5201-05 (1980)); Colony blots (Grunstein et al., Proc. Nat'l Acad. Sci. USA, 72:3961-65 (1975), which are hereby incorporated by reference). Alternatively, the isolated DNA molecule of the present invention can be used in a gene amplication detection procedure (e.g., a W095/06726 ~ PCT~S91 polymerase chain reaction). See H.A. Erlich et. al., "Recent Advances in the Polymerase Chain Reaction", Science 252:1643-51 (1991), which is hereby incorporated by reference.
More generally, the molecular basis for the-uptake phenomenon achieved by Mycobacterium tuberculosis can be utilized to effect uptake of other materials into mammalian cells. This is achieved by utilizing the protein or polypeptide of the present invention in association with such materials for uptake by mammalian cells. This phenomenon can be used to introduce a wide variety of materials into such cells, including antibiotics, DNA
fragments, anti-neoplastic agents, and mixtures thereof.
The opportunity for direct cell entry of antibiotics constitutes a substantial advance, because they will be able to kill intracellular Mycobacterium tuberculosis. One approach for achieving such uptake is by impregnating microspheres with antibiotics and then coating the spheres with the protein or polypeptide of the present invention in order to achieve such uptake. Alternatively, instead of utilizing microspheres to transport antibodies, such therapeutics can be chemically linked to the protein or polypeptide of the present invention.
This technology can be used to treat a wide array of diseases caused by intracellular pathogens. For treatment of tuberculosis, a repertoire of antibiotics, having themselves poor cellular penetration but high activity against extracellular Mycobacterium tuberculosis when tested in vitro, can be utilized in conjunction with the protein or polypeptide of the present invention. In cancer treatment, intracellular delivery of anti-neoplastic agents can be greatly enhanced by conjugating such agents to the protein or polypeptide of the present invention. This will enable reductions in dosages for such agents and in their resulting toxicity.

W095/06726 l q8 PCT~S94/09863 Another aspect of the present invention is to utilize the protein or polypeptide of the present invention in gene therapy or in a genetic vaccine where pieces of therapeutically or prophylactically useful DNA are - 5 conjugated at their thymine residues to the protein or polypeptide of the present invention via linker arms. As a result, genetic material can be introduced into cells to correct genetic defects or to produce a desired characteristic or products that serve as immunogens.
EXAMPLES

ExamPle 1 - Preparation of and Screening for HeLa Cell Invasion Clones To identify the Mycobacterium tuberculosis DNA
sequence that encode m~mm~l ian cell entry, recombinant invasive clones were constructed as follows: Mycobacterium tuberculosis H37Ra strain (ATCC 25177) genome was digested with restriction enzymes Sau3 A1 and Eco R1, and the DNA
fragments were ligated into the Bam H1-Eco R1 restriction sites of a phagemid vector pBluescript II (Stratagene, La Jolla, CA). The recombinant vectors were introduced into E.
coli EL1-Blue (Stratagene) by electroporation. We screened the recombinant strains for HeLa cell-invasive clones by a method similar to that described by R.R. Isberg and S.
Falkow, Nature 317, 262 (1987), which is hereby incorporated by reference.
One E. coli transformant XL1-Blue(pZX7), which harbored a plasmid (pZX7) containing a 1535-base insert in the Bam HI-Eco RI restriction enzyme sites of the pBluescript vector, was found by the screening procedure to associate consistently with HeLa cells. It was confirmed by transmission electron microscopy that this clone entered HeLa cells (Fig.l). Figure lA shows HeLa cells infected W095/06726 2 17 0 1 4 ~ PCT~S94109863 with Mycobacterium tuberculosis strain H37Ra (ATCC 25177), while the invasive recombinant strain E. coli XL1-Blue(pZX7) is shown in Figures lB and lC. The cells were incubated with Mycobacterium tuberculosis strain for 72 hours in Figure lA and with XL1-Blue(pZX7) for 7.5 hours in Figure lB
and Figure lC. Internalization of this clone by HeLa cells was time-dependent (Fig. lB), with intracellular organisms visible as early as 3.5 hours after infection. Some phagosomes contained multiple organisms (Fig. lC), which suggested that the bacteria proliferated intracellularly.
Some of the internalized bacilli were surrounded by a distinct ETZ, similar in appearance to the clear zone surrounding Mycobacterium tuberculosis inside HeLa cells (Fig. lA, arrow). Whether this zone represents the ETZ
often seen around other pathogenic intracellular my-cobacterial organisms (See P. Draper and R.J.W. Rees, Nature 228, 860 (1970); N. Rastogi, Res. Microbiol. 141, 217 (1990); T. Yamamoto, M. Nishimura, N. Harada, T. Imaeda, Int. J. Lepr. 26, 111 (1958), which are hereby incorporated by reference) or is an artifact of the preparation is not clear.
Nonpathogenic E. coli XL1-Blue strains containing the vector pBluescript or another pBluescript-derived recombinant vector (pZN7) showed no association with HeLa cells after 7.5 hours.
To demonstrate that the invasive phenotype was indeed encoded by the cloned Mycobacterium tuberculosis DNA
fragment, we transformed other nonpathogenic E. coli strains, specifically, HB101, DH5~, and NM522, with pZX7.
The constructs HBlOl(pZX7), DH5~(pZX7), and NM522(pZX7) were invasive for HeLa cells. A spontaneous loss of pZX7 on prolonged storage of XL1-Blue(pZX7) was associated with loss of the invasive phenotype.
Four exonuclease III unidirectional deletion subclones of pZX7 and the subclones Bam HI-Pst I (pZX7.1), W095/06726 ~ 8 PCT~S94/09863 Pst-I-HinD III (pZX7.2), and Bam HI-Eco RI ([Zx7.7) was utilized for HeLa cell association. The unidirectional deletion subclones of pZX7 were generated using exonuclease III according to the manufacturer's instruction (Erase-a-Base System, Promega, Madison, WI). The plasmid pZX7 wasdouble-digested with HinD III and Kpn I restriction enzymes downstream from the Eco RI site of the Ban HI-Eco RI DNA
insert to generate a 5' protruding end adjacent to the insert and a four-base 3' protruding end adjacent to the insert and a four-base 3' protrusion at the opposite strand to protect it from Exo III digestion. The digested plasmid was mixed with 300 U of Exo III at 37C, and every 30 s

2.5~1 aliquots of the Exo III digestion were transferred to tubes containing S1 nuclease to remove the remaining single-stranded tails. The S1 nuclease was inactivated byneutralization and heating at 70C for 10 min. Klenow DNA
polymerase was added to create blunt ends which were ligated to circularize the deletion-containing vectors. The ligation mixture was then used to transform the competent E.
coli XL1-Blue strain by electroporation. These transformed strains were incubated for 6 hours with a HeLa cell monolayer.
The results of this procedure are shown in Figure 2. The black bars represent the Mycobacterium tuberculosis DNA sequences, and the white bars represent pBluescript sequences. As shown, the strains of E. coli XLl-Blue harboring pZX7.3, pZX7.4, or pZX7.5 associated with HeLa cells in a pattern similar to that for E. coli ZL1-Blue(pZX7), whereas the other subclones did not.
Example 2 - Infection of Human Macrophages Macrophage monolayers infected with the E. coli recombinant clones of Example 1 were established on glass cover slips at the bottom of polystyrene wells. They were WO 95/06726 PCT/US91i'09 ~
217 01~8 --initially infected with ~ 10 over-night-growth bacteria per macrophage cell for 1 or 2 hours followed by washing with phosphate-buffered saline (pH 7.4) and incubation for an additional 1, 6, or 22 hours. Cultures were performed at 37C in RPMI-1640 medium (Gibco) with 2~ AB heat-inactivated human serum containing gentamicin (10 ~g/ml). The gentamicin was included to kill the extracellular bacteria.
The macrophage monolayer was washed again and then lysed with sterile, distilled water. The lysate was plated on tryptic soy agar medium to obtain colony counts. For microscopy, the macrophage monolayer was fixed with 100~
methanol, stained with 10~ Giemsa stain, and ex~m'ned by light microscopy or processed for electron microscopy.
The monolayer that was infected for 1 hour only was l~m;ned by light microscopy immediately after it was washed, fixed, and stained. The macrophage lysate culture and light microscopy results are shown in Table 1, infra.
The percentage of infected macrophages was calculated from counts or infected macrophages per 100 to 200 macrophage cells on a cover slip monolayer. Each E. coli strain was tested four to six times for each time point, and the means of the percentages of the cells infected by the E. coli recombinant clone and the control strains XL1-Blue(pBluescript) and XL1-Blue(pZX7.3) were compared by students T test.
Figure 3 shows thin-section electron micrographs of human macrophages exposed to the invasive recombinant E.
coli clone XL1-Blue(pZX7) for 3 hours (Figure 3A) and 24 hours (Figure 3B). In Figure 3C, the thin-section micrograph is of human macrophages exposed to nonpathogenic E. coli XL1-Blue(pBluescript) for 24 hours. After 24 hours, bacilli were more numerous inside the cells, compartmentalized, surrounded by multiple layers of a membrane presumably of host origin (Fig. 3B). No bacteria W095106726 7l ~$ PCT~S91~ 3 could be seen inside macrophages infected with E. coli (pBluescript) after 24 hours (Figure 3C).
Table 1 shows the results obtained from this light microscopy and culture study of human macrophage monolayer cells infected with the HeLa cell-invasive E. coli XL1-Blue (pZX7), subclone XL1-Blue (pZX7.3), and noninvasive XL1-Blue (p. Bluescript). The colony-forming units (CFU) were determined per milliliter of cell culture lysate. As shown, after 1 hour of infection, the percentage of cells infected by the recombinant clone (82 + 8~) was more than five times that of cells infected by XL1-Blue(pBluescript) (15 + 6~, P
< O . 001) .
Table 1 Percentage of infected cells (mean _ SEM) CFU per milliliters of lysate Exposure Culture (mean + SEM) (hours) pBluescript pZX7.3 pZX7pBluescript pZX7 115 + 6 59 + 10** 82 + 8**** ND***** ND

3 9 + 4 ND 55 + 171800 + 500 3500 + 1700 8 4 + 2 ND 35 + 510 + 5 1600 + 400 2412 + 10 23 + 8* 60 + 13*** 3 + 1 1300 + 200 *P ~ 0.05, compared with pBluescript clone. 0.001, compared with pBluescript or pZX7.3 clones. 0.05 compared with pZX7.3 clone.
**P < 0.001, compared with pBluescript clone.
***P < 0.001, compared with pBluescript or pZX7.3 clones.
****P < 0.0001, compared with pBluescript clone, P ~ 0.05 compared with pZX7.3 clone.
*****ND means not determined.

This observation suggests that the cloned Mycobacterium tuberculosis DNA sequences facilitate bacterial uptake at quantities above the background phagocytic activity of the macrophage cells. After 24 hours of infection, 12~ (+ 10~) of the macrophages exposed to XL1-Blue(pBluescript) and 60~
(+ 13~) of the cells exposed to XL1-Blue(pZX7) were infected W095/06726 PCT~S94/09863 ~l~ol4s (P < 0.001). As demonstrated in Table 1, culture of the lysate of macrophages that had been infected for 24 hours showed that the intracellular E. coli XL1-Blue(pZX7) strains were viable.
In comparing capacity of XL1-Blue(pZX7), XL1-Blue(pBluescript), and one HeLa cell-invasive deletional derivative, E. coli XL1-Blue(pZX7.3), to infect macrophages from Table 1, at 1 hour of infection, the invasive capacity of E. coli XL1-Blue(pZX7.3) was four times that of XL1-Blue(pBluescript) (P ~ 0.001), but by 24 hours the difference was no longer apparent. Thus, the DNA sequences associated with HeLa cell invasion are responsible for increased uptake by the macrophage, and the sequences that confer survival within the macrophage are located downstream of those necessary for mAmm~lian cell entry.

Example 3 - Homology Analysis The Bam Hi-Eco Ri DNA fragment was sequenced by the chain termination method, described in F. Sanger, et.
al., "DNA Sequencing with Chain-Terminating Inhibitors,"
Proc. Nat. Acad. Sci., 74:5463-67, which is hereby incorporated by reference, and found to have 1535 base pairs [European Molecular Biology Laboratory (EMBL) accession number X70901]. The sequence showed no homology with any of the DNA sequences in the database of GenBank (R72.0) or EMBL
(R31.0). No obvious procaryotic promoter consensus sequence could be discerned. If we assume that Mycobacterium tuberculosis uses the common prokaryotic termination codon sequences, amino acid sequence homologies can be identified.
A region near the NH2-terminus of the deduced sequence of one potential open reading frame was found to share (i) 27 identity with an 80-residue NH2-terminus region of internalin, a protein encoded by Listeria monocytogenes that is associated with mammalian cell entry (A.B. Hartman, M.

W095/06726 ~0 t PCT~S~ E3 Venkatesan, E.V. Oaks, J.M. Buysse, J. Bacteriol, 172, 1905 (1990), which is hereby incorporated by reference); (ii) 20 identity with a 145-residue region of the IpaH gene product of the invasiveness plasmid of Shigella (B. E. Anderson, G.A.
McDonald, D.C. Jones, R.L. Regnery, Infect. Immun. 58, 2760 (1990), which is hereby incorporated by reference); and (iii) 18~ identity with a 176-residue region of human ~-adaptin, a plasma membrane protein that links clathrin to receptors in coated vesicles which are responsible for receptor-mediated endocytosis (S. Ponnambalam, M.S.
Robinson, A.P. Jackson, L. Peiper, P. Parham, J. Biol. Chem.
265, 4814 (1990) and J.L. Goldstein, M.S. Brown, R.G. W.
Anderson, D.W. Russell, W.J. Schneider, Annu. Rev. Cell Biol. 1,1 (1985), which are hereby incorporated by reference). When aligned against the invasin protein of Yersinia pseudotuberculosis, the region associated with cell entry was 19~ identical with a 100-residue region near the invasion COOH-terminus (R.R. Isberg, D.L. Voorhis, S.
Falkow, Cell 50, 769 (1987), which is hereby incorporated by reference). The functional significance of these alignments is not clear.

ExamPle 4 - Functional Analysis of 52kD Polypeptide Protein fractions analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared as follows: A
5-ml aliquot of bacterial overnight growth (adjusted to absorbance at 550 nm at optical density 600) in tryptic soy broth containing ampicillin (100 ~g/ml) was harvested by centrifugation. We then sonicated the bacterial pellet in 1.5 ml of 10 mM tris-HCI buffer (pH 8.0) containing 5 mM
MgCI2. The sonicate was centrifuged for 25 min at 12,000 rpm in a microcentrifuge (Eppendorf model 5415C) at 4C.
Acetone was added to 600 ~l of the supernatant in a fresh microcentrifuge tube (60~ v/v), and the mixture was W095/06726 2 17 0 1 ~ 8 PCT~S~ g~S~

centrifuged for 25 min. at 14,000 rpm at 4C. The pellet was resuspended in 20 ~l of distilled water and 20 ~l of Laemmli's boiling buffer, heated over boiling water for 5 min. and analyzed by SDS-PAGE. The bacterial debris containing the outer membrane fraction after the first centrifugation was resuspended in 100 ~l of water and 100 ~l of 15 mM tris-HCI buffer (pH 8.0) containing 7.5 mM MgCI2 and 3~ (v/v) Triton X-100 and centrifuged for 25 min. at 14,000 rpm. The pellet was resuspended in 25 ~l of water and 25 ~l of boiling buffer and boiled it and analyzed a 20-~l aliquot of the sample by SDS-PAGE.
The SDS-PAGE (i.e., SDS-polyacrylamide gel electrophoresis) of acetone precipitated a soluble fraction of bacterial cell sonicate. The polypeptides were analyzed in a 9~ gel (left): molecular size standards (lane 1), E.
coli XL1-BBlue with a vector (pZN) containing an unrelated Mycobacterium tuberculosis DNA fragment between the Bam HI-Eco RI pBluescript cloning sites (lane 2), and XL1-Blue(pZX7) (land 3). Analysis in an 8~ gel (right): XL1-Blue containing a vector (pZX7.8) with a two base frameshiftintroduced 12 bases upstream from the Bam HI cloning site in pZX7 (lane 1) and XL1-Blue(PZX7) (lane 2). Molecular sizes are indicated at the far right. We detected a 52-kD
polypeptide in the soluble protein fraction of XL1-Blue(pZX7) (arrow). A protein of about 50 kD is expressedby XL1-Blue containing pZX7.8. The expression of the 52-kD
protein was always associated with HeLa cell interaction of the recombinant E. coli clone.
From the SDS-PAGE results of Figure 4, it can be concluded that a soluble fraction of the bacterial cell sonicate of XL1-Blue(pZX7) contained a 52-kD polypeptide that was not detected in the soluble fraction of XL1-Blue with a pBluescript-derived vector (pZN7) harboring an unrelated Mycobacterium tuberculosis DNA fragment. A two-base frameshift, introduced by blunt-end ligation after the WO95/06726 1 7l ¦8 PCT~S9~/09~3 5~ protruding end had been filled with Klenow DNA polymerase at the Xba I site 12 bases upstream from the Bam HI cloning site in pZX7 (confirmed by sequencing), led to loss of association with HeLa cells of the E. coli XLl-Blue containing this plasmid (pZX7.8). This clone did not express the 52-kD protein, but a new polypeptide of lower molecular mass was detected in the soluble fraction. A
spontaneous loss of the capacity to associate with HeLa cells after prolonged storage of XLl-Blue(pZX7) was accompanied by loss of the 52-kD protein. Hence, this 52-kD
protein is likely to be a product expressed by the cloned Mycobacterium tuberculosis DNA fragment. There were no detectable differences in the bacterial outer membrane polypeptide fractions.
It is not clear if the cloned 1535-bp fragment has more than one open reading frame or if a single gene product mediates both cell invasion and survival inside the macrophage. Drugs designed to target an Mycobacterium tuberculosis product mediating macrophage survival, or vaccines against a product encoding mammalian cell entry, could contribute substantially to worldwide tuberculosis control strategies.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims (52)

WHAT IS CLAIMED:

1. An isolated DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

2. An isolated DNA molecule according to claim 1, wherein said DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 1.

3. An isolated DNA molecule according to claim l, wherein said DNA molecule encodes for a polypeptide having a molecular weight of about 50-55 kilodaltons.

4. An isolated protein or polypeptide encoded by a DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

5. An isolated protein or polypeptide according to claim 4, wherein said DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 1.

6. An isolated protein or polypeptide according to claim 5, wherein the protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 2.

7. An isolated protein or polypeptide according to claim 4, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

8. An isolated protein or polypeptide according to claim 4, wherein said protein or polypeptide is recombinant.

9. An isolated protein or polypeptide according to claim 4, wherein said protein or polypeptide is purified.

10. An isolated protein or polypeptide according to claim 4, wherein said protein or polypeptide has one or more antigenic determinants conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

11. A method of vaccinating mammals against infection by Mycobacterium tuberculosis comprising:
administering an effective amount of the isolated protein or polypeptide according to claim 4 to mammals.

12. A method according claim 11, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

13. A recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

14. A recombinant DNA expression system according claim 13, wherein said heterologous DNA comprises the nucleotide sequence corresponding to SEQ. ID. No. 1.

15. A recombinant DNA expression system according to claim 13, wherein said heterologous DNA is inserted into said vector in proper orientation and correct reading frame.

16. A host cell incorporating a heterologous DNA
conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

17. A host cell according to claim 16, wherein said heterologous DNA comprises the nucleotide sequence corresponding to SEQ. ID. No. 1.

18. A host cell according to claim 16, wherein said heterologous DNA is inserted in a recombinant DNA
expression system comprising an expression vector.

19. A vaccine for preventing infection and disease of mammals by Mycobacterium tuberculosis comprising:
an isolated protein or polypeptide according to claim 4; and a pharmaceutically-acceptable carrier.

20. A vaccine according to claim 19, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

21. A vaccine according to claim 19, wherein said protein or polypeptide is purified.

22. A method of vaccinating mammals against infection by Mycobacterium tuberculosis comprising:
administering an effective amount of the vaccine according to claim 19 to mammals.

23. A method according claim 22, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

24. An isolated antibody or binding portion thereof against a protein or polypeptide according to claim 4.

25. An isolated antibody or binding portion thereof according to claim 24, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

26. An isolated antibody or binding portion thereof according to claim 24, wherein said antibody is monoclonal or polyclonal.

27. An isolated antibody or binding portion thereof according to claim 24, wherein said antibody is specific for an antigenic determinant of said protein or polypeptide encoded by a gene fragment conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

28. A method of passively immunizing mammals infected with Mycobacterium tuberculosis comprising:
administering an effective amount of said antibody or binding portion thereof according to claim 24 to mammals-infected with Mycobacterium tuberculosis.

29. A method according to claim 28, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

30. A composition for passively immunizing mammals infected with Mycobacterium tuberculosis comprising:
an isolated antibody or binding portion thereof according to claim 24; and a pharmaceutically-acceptable carrier.

31. A composition according to claim 30, wherein said antibody is monoclonal or polyclonal.

32. A composition according to claim 30, wherein said antibody is specific for an antigenic determinant of said protein or polypeptide encoded by a gene fragment conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.

33. A method of passively immunizing mammals infected with Mycobacterium tuberculosis comprising:
administering an effective amount of said composition according to claim 30 to mammals infected with Mycobacterium tuberculosis.

34. A method according to claim 33, wherein said administering is oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal.

35. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising:
providing a protein or polypeptide according to claim 4 as an antigen;
contacting the sample with the antigen; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample using an assay system.

36. A method according to claim 35, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

37. A method according to claim 35, wherein the assay system is selected from the group consisting of an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.

38. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising:
providing an antibody or binding portion thereof according to claim 24;
contacting the sample with the antibody or binding portion thereof; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample using an assay system.

39. A method according to claim 38, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

40. A method according to claim 38, wherein the assay system is selected from the group consisting of an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, and an immunoelectrophoresis assay.

41. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising:
providing a nucleotide sequence of the DNA
molecule according to claim 1 as a probe in a nucleic acid hybridization assay;
contacting the sample with the probe; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample.

42. A method for detection of Mycobacterium tuberculosis in a sample of tissue or body fluids comprising:
providing a nucleotide sequence of the DNA
molecule according to claim 1 as a probe in a gene amplification detection procedure;
contacting the sample with the probe; and detecting any reaction which indicates that Mycobacterium tuberculosis is present in the sample.

43. A product for uptake of materials into mammalian cells comprising:
a material for uptake by mammalian cells; and a protein or polypeptide according to claim 4, wherein said protein is associated with said material.

44. A product according to claim 43, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

45. A product according to claim 43, wherein said protein or polypeptide is purified.

46. A product according to claim 43, wherein said material is selected from the group consisting of antibiotics, DNA fragments, anti-neoplastic agents, and mixtures thereof.

47. A cellular uptake process comprising:
directing a material into mammalian cells with a protein or polypeptide according to claim 4.

48. A process according to claim 47, wherein said protein or polypeptide is a polypeptide having a molecular weight of about 50-55 kilodaltons.

49. A process according to claim 47, wherein said protein or polypeptide is purified.

50. A process according to claim 47, wherein said material is selected from the group consisting of antibiotics, DNA fragments, anti-neoplastic agents, and mixtures thereof.

51. A process according to claim 47, wherein the mammalian cells are macrophages.

52. A process according to claim 51, wherein said process induces cell-mediated immunity.