# Patent

## Title
Methods for detection of mycobacteria

## Abstract

Nucleic acid encoding four novel immunodeterminant protein antigens of M. bovis BCG, which is a vaccine strain for tuberculosis, have been isolated. These genes were isolated as immunoreactive recombinant clones from a genomic library of M. bovis BCG DNA and screened with sera collected from tuberculosis patients. The BCG DNA expressed an antigen of Mr 90 kD, and an ORF encoding 761 amino acids encoding a protein of deduced molecular weight 79 kD, was indentified to encode a membrane bound, ion-motive ATPase of M. bovis BCG. The approach described here can be used to identify immunogens of mycobacteria. The well-characterized M. bovis BCG antigens can be used in the prevention, diagnosis and treatment of tuberculosis. The 79 kD antigen is also useful in the design of recombinant vaccines against different pathogens. The sequence of the 79 kD membrane-associated polypeptides also are useful for the development of specific PCR amplification based diagnostic procedures for the detection of mycobacteria. The promoter of the 79 kD antigen is useful for expressing homologous and/or heterologous antigens in mycobacteria.

## Background
This is a continuation of application Ser. No. 09/099,902, filed on Jun. 18, 1998, which is a divisional of application Ser. No. 08/710,676, filed Sep. 23, 1996, now issued as U.S. Pat. No. 5,770,719 which is a divisional of application Ser. No. 08/192,632, filed on Feb. 7, 1994, now issued U.S. Pat. No. 5,559,011; which is a divisional of application Ser. No. 07/906,395, filed on Jun. 29, 1992, now issued U.S. Pat. No. 5,330,754.


TECHNICAL FIELD OF THE INVENTION
The invention relates to membrane-associated polypeptides of mycobacteria and, in particular, the use of such polypeptides and the nucleic acids encoding them for use as vaccines and diagnostic reagents.
BACKGROUND OF THE INVENTION
The mycobacteria are a diverse collection of acid fast, gram-positive bacteria, some of which cause important human and animal diseases. In humans, the two most common mycobacteria-caused diseases are tuberculosis (TB) and leprosy, which result from infection with M. tuberculosis and M. leprae, respectively.
Tuberculosis displays all of the principal characteristics of a global epidemic disease. Currently, tuberculosis afflicts more than 35 million individuals worldwide and results in over 4 million deaths annually. In India, at any given time, almost 8 million people are reported to suffer from this disease and 500,000 deaths recorded. These figures may not cover the totality of those suffering from this disease in this country. Thus, tuberculosis appears to be a problem of major concern in India as also in many other countries of the world.
Tuberculosis is caused by M. tuberculosis, M. bovis, M. africanum and M. microti, the acid-fast, Gram positive, tubercle bacilli of the family Mycobacteriaceae. Some local pathogenic strains of M. tuberculosis have also been isolated from patients in Madras and other cities in India, which differ in some respects from M. tuberculosis H37Rv, which is a virulent strain.
In recent years, certain groups of individuals with AIDS have been found to have a markedly increased incidence of TB as well. It has now been shown that one group of mycobacteria which consists of M. avium, M. intracellulare and M. scrofulaceum, jointly known as MAIS complex, is responsible for disseminated disease in a large number of persons with AIDS (Kiehn et al., J. Clin. Microbiol., 21:168-173 (1985); Wong et al., Amer. J. Med., 78:35-40 (1985)).
Since Koch identified M. tuberculosis as the causative agent of tuberculosis in 1882, many scientific studies and public health efforts have been directed at diagnosis, treatment and control of this disease. However, characteristics of M. tuberculosis have hampered research to improve diagnosis and to develop more effective vaccines. In addition, the biochemical composition of the organism has made identification and purification of the cellular constituents difficult, and many of these materials once purified, lack sensitivity and specificity as diagnostic reagents. As a result, diagnostic and immunoprophylactic measures for mycobacterial diseases have changed little in the past half century. The conventional methods for the diagnosis of M. tuberculosis are troublesome and results are delayed.
Bacillus Calmette-Guerin (BCG), an avirulent strain of M. bovis (Calmette, A., Masson et Cie, Paris (1936)), is used extensively as a vaccine against tuberculosis. Though numerous studies have found that it has protective efficacy against tuberculosis (Luelmo, F., Am. Rev. Respir. Dis., 125, 70-72 (1982)) BCG has failed to protect against tuberculosis in several trials (WHO, Tech. Rep. Ser., 651:1-15 (1980)) for reasons that are not entirely clear (Fine, P., Tubercle, 65:137-153 (1984); Fine, et al., Lancet, (ii):499-502 (1986)).
The eradication with vaccination, early diagnosis, and efficient therapy is an important objective of the drive to combat mycobacterioses. The lacunae in the present knowledge of the biology of these pathogens—their make-up, their natural history, their physiology, biochemistry and immunological reactivities, highlights the need for attempts to unravel their weaknesses, so that more efficient ways to combat this disease can be devised. To develop more effective tools for the diagnosis and prevention of these diseases, it is important to understand the immune response to infection by mycobacterial pathogens. The mycobacterial components that are important in eliciting the cellular immune response are not yet well defined. The antibody and T-cell responses to infection or inoculation with killed mycobacteria have been studied in humans and in animals. Human patients with TB or leprosy produce serum antibodies directed against mycobacterial antigens. Although antibodies may have some function in the antimycobacterial immune response, the exact function remains to be clarified since no protective role can be ascribed to these antibodies. Protection against mycobacterial diseases involves cell-mediated immunity.
Mycobacteria do not produce any directly toxic substances and consequently their pathogenicity results from multiple factors involved in their interaction with the infected host. Intracellular parasitism probably depends on host cell trophic factors; it is conceivable that their short supply may be bacteriostatic and could play a role in the mechanism of mycobacterial dormancy.
It is generally understood that protective immunity in mycobacterial infection is mediated by specific T cells which activate macrophages into non-specific tuberculocidal activity. Evidence suggests that gamma-INF triggers macrophages towards H2O2-mediated bacterial killing, but related or other macrophage activating factor (MAF) molecules may also be involved. The causes responsible for the inadequate bactericidal function at sites of abundant T cell proliferation have not yet been explained. Dissociation between delayed-type hypersensitivity (DTH) and protective immunity led to views that T-cells of a distinct subset or specificity could be responsible for the acquired resistance to mycobacterial infection. Alternatively, interference with protection may result from corollary cellular reactions, namely by suppressor T-cells and macrophages, or from the shifting of T-cells towards helper function for B-cells.
Unlike viral and some parasite pathogens which can evade host resistance by antigenic shift, mycobacteria have a resilient cell wall structure and can suppress host immune responses by the action of their immunomodulatory cell wall constituents. Whilst the success of protective immunization towards other microbial pathogens mainly depends on quantitative parameters of immunity, it appears that mycobacterial immunomodulatory stimuli produce a regulatory dysfunction of the host immune system. This may not be possible to override simply by more resolute immunization using vaccines of complex composition such as whole mycobacteria (e.g. BCG). Perhaps mycobacteria did not evolve potent “adjuvant” structures to boost the host immunity but rather to subvert host defenses towards ineffective cellular reactions operating to the advantage of the pathogen. Vaccination with an attenuated pathogen such as BCG could amplify further immune responses but with limited protection of the host, the potential scope for immunization with defined antigens is yet to be explored.
The purification and characterization of individual antigenic proteins are essential in understanding the fundamental mechanism of the DTH reaction on the molecular level. The possible functional role of proteins of defined structure in the pathogenesis of mycobacterial diseases as well as for diagnostic purposes remains of great interest. Numerous groups have attempted to define mycobacterial antigens by standard biochemical and immunological techniques, and common as well as species specific antigens have been reported in mycobacteria (Minden, et al., Infect. Immun., 46:519-525 (1984); Closs, et al., Scand. J. Immunol., 12:249-263 (1980); Chaparas, et al., Am. Rev. Respir. Dis., 122:533 (1980); Daniel, et al., Microbiol. Rev., 42:84-113 (1978); Stanford, et al., Tubercle, 55:143-152 (1974); Kuwabara, S., J. Biol. Chem., 250:2556-2562 (1975)).
Very little information about the mycobacterial genome is available. Initially, basic studies were conducted to estimate the genome size, G+C content and the degree of DNA homology between the various mycobacterial genomes (Grosskinsky, et al., Infect. Immun., 57, 5:1535-1541 (1989); Garcia, et al., J. Gen. Microbiol., 132:2265-2269 (1986); Imaeda, T., Int. J. Sys. Bacteriol., 35, 2:147-150 (1985); Clark-Curtiss, et al., J. Bacteriol., 161 3:1093-1102 (1985); Baess, I. et al., B., Acta. Path. Microbiol. Scand., (1978) 86:309-312; Bradley, S. G., Am. Rev. Respir. Dis., 106:122-124 (1972)). Recently, recombinant DNA techniques have been used for the cloning and expression of mycobacterial genes. Genomic DNA fragments of M. tuberculosis, M. leprae and some other, mycobacterial species were used for the construction of lambda gtll phage (Young, et al., Proc. Natl. Acad. Sci., U.S.A., 82:2583-2587 (1985); Young, et al., Nature (London), 316:450-452 (1985)) or other vector-based recombinant gene libraries. These libraries were screened with murine monoclonal antibodies (Engers, et al., Infect. Immun., 48:603-605 (1985); Engers, et al., Infect. Immun., 51:718-720 (1986)) as well as polyclonal antisera and some immunodominant antigens were identified. The principal antigen among these being five 12, 14, 19, 65 & 71 kDa of M. tuberculosis (Young et al., Proc. Natl. Acad. Sci., U.S.A., 82:2583-2587 (1985); Shinnick et al., Infect. Immun., 55(7):1718-1721 (1987); Husson and Young, Proc. Natl. Sc. Acad., 84:1679-1683 (1987); and five 12, 18, 23, 36 & 65 kDa antigens of M. leprae (Young, et al., Nature (London), 316:450-452 (1985)). A few homologues of some of these antigens were also identified in some other mycobacterial species (e.g., BCG) (Yamaguchi et al., FEB 06511, 240:115-117 (1988); Yamaguchi et al., Infect. Immun., 57:283-288 (1989); Matsuo, et al., J. Bacteriol., 170, 9:3847-3854 (1988); Radford, et al., Infect. Immun., 56, 4:921-925 (1988); Lu, et al., Infect. Immun., 55, 10:2378-2382 (1987); Minden, et al., Infect. Immun., 53, 3:560-564 (1986); Harboe, et al., Infect. Immun., 52, 1:293-302 (1986); Thole, et al., Infect. Immun., 50, 3:800-806 (1985)). These antigens, however, are either intracellular or secreted molecules.
Although M. bovis BCG has been widely used as a vaccine against tuberculosis, the determination of the membrane-associated polypeptides of mycobacterium that are capable of inducing a protective immune response is highly desirable. The use of such a membrane-associated polypeptide or the DNA encoding it provides for the generation of recombinant vaccines, e.g., mycobacterial membrane-associated immunogens expressed in, for example, a virus or bacterium such as vaccinia virus, Salmonella, etc. used as a live carrier, or the display of non-mycobacterial immunogens on the surface of a cultivable mycobacterial strain which can be used as a live recombinant vaccine.
Accordingly, it is an object herein to provide methods for identifying and isolating nucleic acids encoding a membrane-associated polypeptide of mycobacteria.
Further, it is an object herein to provide membrane-associated polypeptides of mycobacteria and the nucleic acids encoding it.
Still further, it is an object herein to provide vaccines utilizing all or part of the membrane-associated polypeptide of a mycobacterium or the DNA encoding such membrane-associated polypeptide.
Still further, it is an object to provide reagents comprising said membrane-associated polypeptide with a mycobacterium or DNA encoding it useful in diagnostic assays for mycobacterial infection.
Still further, it is an object to provide a promoter sequence comprising the promoter of said membrane associated polypeptide, which can direct gene expression in mycobacteria as well as in other microorganisms such as E. coli. 
SUMMARY OF THE INVENTION
In accordance with the foregoing objects, the invention includes compositions comprising nucleic acid encoding all or part of a membrane-associated polypeptide of a mycobacterium and the membrane-associated polypeptide encoded by said DNA. The membrane-associated polypeptide is characterized by the ability to detect an immune response to pathogenic mycobacteria or the mycobacteria from which the membrane associated polypeptide or part thereof is derived. Such mycobacteria include M. bovis, M. tuberculosis, M. leprae, M. africanum and M. microti, M. avium, M. intracellular and M. scrofulaceum and M. bovis BCG.
A particular mycobacterial membrane-associated polypeptide is a 79 kD ion-motive ATPase. Extra-cellular, intra-cellular and transmembrane domains are identified in this mycobacterial membrane-associated polypeptide based upon its DNA and deduced amino acid sequence.
The invention also includes vaccines utilizing all or part of a membrane-associated mycobacterial polypeptide or an expressible form of a nucleic acid encoding it. The invention also includes mycrobacterial promoter sequences capable of directing gene expression in mycobacteria as well as in other microorganisms such as E. coli. Such promoters are from mycobacterial genes encoding membrane-associated ATPases. A preferred promoter is that of the gene encoding the M. bovis BCG 79 kD membrane-associated polypeptide. This promoter sequence is especially useful to express genes of interest in mycobacteria.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the results of immunoscreening of recombinant colonies carrying M. bovis BCG DNA (panel A) and M. tuberculosis H37Rv DNA (panel B), using sera from TB patients in which the presence of M. bovis BCG antigens and M. tuberculosis H37Rv antigens capable of reacting with the antisera is indicated by a qualitative signal.
FIG. 2 shows the comparison of restriction site maps of recombinant clones carrying BCG DNA identified using the immunoscreening assay described herein (panel B) with the restriction site maps of five immunodominant antigens of M. tuberculosis and M. bovis BCG genomic DNAs, respectively, (Husson and Young, Proc. Natl. Acad. Sci., U.S.A., 84:1679-1683 (1987); Shinnick et al., Infect. Immun., 55:1718-1721 (1987) (panel A)). Restriction maps in each panel have been drawn to the same scale (indicated at the top), and restriction sites are indicated above the restriction maps. The dotted line in panel A represents the non-mycobacterial DNA. Restriction enzymes: B, BamHI, E, EcoRI, G, BglII, K, KpnI, P, PvuI, X, XhoI, H, HincII, U, PvuII, Ps, PstI, Hi, HindIII. In panel A, A is SalI and S is SacI. In panel B, S is SalI.
FIG. 3 illustrates the results of Western blot analysis of the sonicated supernate of recombinant clone pMBB51A which carries a BCG DNA insert identified following immunoscreening of the recombinant colonies. The top panel shows reactivity of MBB51A (lane 2) and E. coli (lane 1) with sera from TB patients. The bottom panel (part A) shows reactivity of MBB51A (lanes 1 and 2) and E. coli (lane 3) with anti-H37Rv sera raised in rabbits. Part B shows reactivity of MBB51A (lanes 1 and 2) and E. coli (lane 3) with the second antibody alone. Arrows indicate the position of the 90 kD immunoreactive BCG protein expressed by the recombinant MBB51A, which was absent in the negative control.
FIG. 4 illustrates the nucleotide sequence (Seq. ID No.: 1) of clone pMBB51A 3.25 kb insert DNA containing the M. bovis BCG immunoreactive MBB51A gene encoding an ion-motive ATPase, with a deduced molecular weight of 79 kD. The deduced amino acid sequence (Seq. ID No.: 2) is shown below the nucleotide sequence. Upstream promoter elements are underlined. Transcription termination region is indicated by inverted arrows. 5′ and 3′ flanking regions are also shown.
FIG. 5 illustrates a schematic model derived for the 79 kD protein encoded by pMBB51A which represents an ion-motive ATPase of BCG. The model considers only the structural and functional features that are prominent in the other ion-motive ATPase homologs of transmembrane domains of the protein. Functionally, important amino acid residues are indicated (P), proline at position 400; (D), aspartic acid at position 443; (G), glycine at position 521; and (A), alanine at position 646. Numbers indicate amino acid residues broadly defining the limits of the transmembrane domains.
FIG. 6 illustrates the results of Southern blot hybridization of BamHI digest of genomic DNAs from M. bovis BCG (lane 6), M. tuberculosis H37Rv (lane 5), M. smegmatis (lane 4) and M. vaccae (lane 3) using pMMB51A DNA insert (lane 8) as probe. Panel A shows ethidium bromide stained gel and panel B shows the results of Southern blot hybridization.



DETAILED DESCRIPTION OF THE INVENTION
As used herein, a “membrane-associated polypeptide” of a mycobacterium is defined as any Mycobacterial membrane-associated polypeptide which is capable of detecting an immune response against the wild-type mycobacterium containing the membrane-associated polypeptide. However, based upon the observed cross-reactivity of the 79 kD membrane-associated polypeptide of an M. bovis BCG with pooled anti-sera from patients afflicted with tuberculosis and the cross-hybridization as between the DNA encoding the 79 kD membrane-associated polypeptide and the DNA of M. tuberculosis H37Rv, the membrane-associated polypeptide of the invention is not limited to that identified herein from M. bovis BCG. Rather, it encompasses not only homologs to the 79 kD ion-motive ATPase but also any and all membrane-associated polypeptides of a mycobacterium that can be used to detect an immune response by the same or a different mycobacteria in which the membrane-associated polypeptide is normally found.
As used herein, “nucleic acid” includes DNA or RNA as well as modified nucleic acid wherein a detectable label has been incorporated or wherein various modifications have been made to enhance stability, e.g., incorporation of phosphorothioate linkages in the phosphoribose backbone, etc. Such nucleic acid also includes sequences encoding the anti-sense sequence of the DNA encoding the membrane-associated polypeptide such that the now well-known anti-sense technology can be used to modulate expression of such membrane-associated polypeptides.
In some aspects of the invention, the nucleic acid sequence encoding all or part of a membrane-associated polypeptide of the mycobacterium is used as a vaccine.
When so-used the nucleic acid is generally an “expressible nucleic acid” that contains all necessary expression regulation sequences to control transcription and translation of the nucleic acid in a designated host system. In some vaccine embodiments, the DNA encodes a chimeric polypeptide containing at least one transmembrane domain of the membrane-associated polypeptide and an “immunogenic polypeptide”. The transmembrane domain is used to display the immunogenic polypeptide on the surface of a particular host organism such as an attenuated live vaccine. When the membrane-associated polypeptide includes more than one transmembrane region, one or more of the transmembrane regions can be used with an immunogenic polypeptide. Thus, for example, the 79 kD ion-motive ATPase as shown in FIG. 5 has at least three extracellular domains into which an immunogenic polypeptide can be engineered by well-known methods involving recombinant DNA technology. Although it is preferred that more than one transmembrane region be used to display an immunogenic polypeptide, one skilled in the art can readily vary the length of such a membrane-associated polypeptide to maximize an immunogenic response or to minimize the amount of membrane-associated polypeptide used in such applications.
As used herein, “immunogenic polypeptide” comprises all or part of any polypeptide which can potentially be utilized in a vaccine or diagnostic application. Thus, the immunogenic polypeptide can comprise heterologous immunogens, i.e., immunogens from non-mycobacterial sources, e.g., Salmonella or Shigella or from different mycobacteria from which the membrane-associated polypeptide is derived, e.g., immunogens from Mycobacterium tuberculosis fused to a membrane-associated polypeptide from M. bovis BCG. However, in some instances homologous immunogens can be used. For example, each of the extracellular domains as set forth in FIG. 5 herein can be combined and displayed by combination with one or more of the transmembrane domains from the membrane-associated polypeptide normally containing them. Alternatively, the intercellular domains can be displayed extracellularly using appropriate transmembrane regions from the same molecule.
In an alternate vaccine embodiment, all or part of the membrane-associated polypeptide of mycobacteria, rather than the DNA encoding, is used as part of a vaccine. Such proteinaceous vaccines are formulated with well-known adjuvants and administered following well-established protocols known to those skilled in the art.
In still other embodiments, the nucleic acid encoding the membrane-associated polypeptide of the invention can be used as a diagnostic for detecting infection based upon hybridization with wild-type genes contained by the infectious mycobacterium. Such detection can comprise direct hybridization of DNA extracted from an appropriate diagnostic sample or PCR amplification using the nucleotide sequence of the nucleic acid encoding the membrane-associated polypeptide of the invention to prime amplification. If PCR amplification is primed in a conserved region the presence of mycobacteria in a diagnostic sample can be determined. If primed in a non-conserved region which is species specific the diagnostic assay determined the specific mycobacterium causing an infection.
In addition, the membrane-associated polypeptide of the invention can also be used to detect the presence of antibodies in the sera of patients potentially infected with mycobacteria. Such detection systems include radioimmunoassays and various modifications thereof which are well-know to those skilled in the art. In addition, the membrane-associated polypeptide of the invention can be used to detect the presence of a cell-mediated immune response in a biological sample. Such assay systems are also well-known to those skilled in the art and generally involve the clonal expansion of a sub-population of T cells responding to stimuli from the membrane-associated polypeptide. When so-used, the humoral and/or cell-mediated response of a patient can be determined and monitored over the course of the disease.
Recombinant clones encoding immunogenic protein antigens of M. bovis BCG have been isolated from a genomic library of M. bovis BCG DNA. In particular, DNA fragments encoding four protein antigens of M. bovis BCG have been isolated by probing a pBR322 library of M. bovis BCG DNA with sera from TB patients, absorbed on E. coli. Restriction site maps of these four recombinant clones are different from those of the five immunodominant antigens of mycobacteria (Young, et al., Proc. Natl. Acad. Sci., U.S.A., 82:2583-2587 (1987); Husson and Young, Proc. Natl. Acad. Sci., U.S.A., 84:1679-1683 (1987); Shinnick et al., Infect. Immun., 55:1718-1721 (1987)), thereby indicating that these cloned protein antigens are novel. One of the recombinant DNA clones encoded an immunoreactive protein with apparent molecular weight of 90 kD as determined by Western blot analysis. The complete nucleotide sequence of the insert DNA of this clone was determined. This clone was found to carry a mycobacterial promoter and a monocistronic ORF encoding a protein of 761 amino acids with a deduced molecular weight of 79 kD. This 79 kD protein had extensive homology with ion-motive ATPases of S. faecalis (Solioz et al., J. Biol. chem, 262:7358-7362 (1987)), E. coli (Hesse et al., Proc. Natl. Acad. Sci., U.S.A., 81:4746-4750 (1984)) and several other organisms, and thus, represents an ion-motive ATPase or a putative K+ATPase of BCG. Using computer algorithms, this ion-motive ATPase was determined to be a membrane protein and has a homologue in M. tuberculosis H37Rv, which is pathogenic in humans, but not in M. vaccae and M. smegmatis, which are non-pathogenic. As a result, novel BCG immunogens can be available which can be useful in the prevention, diagnosis and treatment of tuberculosis and other mycobacterial infections. They can be used, for example, in the development of highly specific serological tests for screening patients for individuals producing antibodies to M. tuberculosis, or those infected with M. tuberculosis, in the development of vaccines against the disease, and in the assessment of the efficacy of the treatment of infected individuals.
Further, based on the nucleotide sequence of the pMBB51A insert DNA, appropriate oligonucleotide primers can be used for PCR amplification using as template M. bovis BCG or M. tuberculosis H37Rv DNA. Such a PCR amplification scheme can be thus useful for the detection of mycobacterial DNA in a given sample. Further, by a judicious choice of the primer design, such an amplification procedure can be adapted for taxonomic classification of mycobacterial DNAs. For example, using primers to flank a heavily conserved region such as the ATP-binding site, PCR amplification is common to all mycobacterial species, whereas using primers from non-conserved areas, amplification can be made species specific.
EXAMPLE I
Isolation and Characterization of Genes Encoding Immogenic Protein Antigens of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37R
A. Construction of Recombinant DNA Libraries of M. bovis BCG DNA and Mycobacterium tuberculosis H37Rv
A recombinant DNA library of M. bovis BCG genomic DNA was constructed using pBR322 a high copy number plasmid vector (Bolivar, et al., Gene, 2:95-113 (1977)) with antibiotic markers (ampicillin and tetracycline) and several unique cloning sites. M. bovis BCG cells were harvested from a culture in late logarithmic phase of growth and high molecular weight DNA was isolated by the procedure of (Eisenach, et al., J. Mol. Biol., 179:125-142 (1986)) with slight modifications. BCG DNA was digested to completion with BamH I and shotgun cloning of these fragments into the BamH I site of pBR322 was performed. The genomic library was transformed into E. coli strain DHI and recombinants were scored on the basis of ampicillin resistance and tetracycline sensitivity. The aim of this approach was to generate restriction fragments of a broad size range so as not to restrict the library to DNA fragments of any particular size range. This cloning strategy also ensured to a large extent that any recombinants selected for expression of mycobacterial antigens should be likely to drive expression from a mycobacterial promoter rather than the Tet promoter of pBR322.
The BCG library constructed in this manner contained 2051 clones of BCG origin. In an analogous manner, a genomic library of Mycobacterium tuberculosis H37Rv DNA was constructed and 1100 clones obtained.
The BCG DNA inserts ranged in size from 0.9 to 9.5 kb. The average size of the mycobacteria DNA fragments inserted in pBR322 was estimated to be about 4 kb. Given the genome size of BCG to be 4.5×103 kb (Bradley, S. G., J. Bacteriol., 113:645-651 (1973); Imaeda, et al., Int. J. Syst. Bacteriol., 32, 456-458 (1982)), about 1000 clones of this average insert size would represent comprehensively the entire genome of the microorganism.
B. Isolation of Recombinant DNA Clones Encoding BCG Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv Protein Antigens
In order to identify recombinants expressing mycobacterial antigens, a colony immunoscreening assay (CIA) to screen recombinant colonies with appropriate antisera, was established. Sera obtained from 20 patients newly diagnosed with active pulmonary tuberculosis were pooled for use in immunoscreening. None of the patients had received treatment for tuberculosis prior to this study and their sputa were positive for acid fast bacteria in all cases. Pooled sera were absorbed on a E. coli sonicate overnight at 4° C., to eliminate antibodies cross-reactive to E. coli antigens, thereby improving signal to noise ratio during the immunoscreening.
Individual recombinant colonies were grown overnight on nitrocellulose membranes and immunoscreening was carried out as described with slight modifications. The colonies were lysed in chloroform vapor to release the cloned mycobacterial antigens, immobilized on the nitrocellulose paper. The immobilized antigens were reacted with TB sera and binding of the antibody was revealed by standard procedures using a horseradish peroxidase-protein A detection system. The signals obtained with the recombinant clones were compared with that obtained in case of E. coli colonies harbouring pBR322 vector alone, which served as the negative control, to assess the signal to noise ratio. Further, to ascertain whether the immunoreactivity of the recombinant clones was due to anti-mycobacterial antibodies or due to a reaction with normal serum components, another CIA of the selected recombinants was performed using TB sera and normal human sera NHS which had been absorbed on E. coli in a manner analogous to that described earlier for TB sera. Only those clones reacting selectively with TB sera and not with NHS, were considered to be unambiguously suggestive of the presence of mycobacterial antigens. The use of this immunoscreening approach to identify recombinant colonies carrying mycobacterial DNA inserts capable of expressing mycobacterial antigens is described below:
FIG. 1 shows the result of immunoscreening of recombinant colonies carrying M. bovis BCG DNA (panel A) or M. tuberculosis H37 Rv DNA (panel B) using sera from TB patients. The colonies were grown on nitrocellulose paper overnight, lysed to release the cloned mycobacterial antigen and allowed to react with the antibodies. The presence of mycobacterial antigen is indicated by a qualitative signal in the recombinant clones which is absent in the negative control comprising colonies harbouring pBR322 vector alone. A similar assay was repeated with normal human serum to ascertain the specificity of the cloned mycobacterial antigens. 51 recombinant colonies carrying M. bovis BCG DNA inserts and 45 recombinant colonies carrying M. tuberculosis H37Rv DNA inserts were screened by the above procedure; 14 clones of BCG origin (panel A) and 2 clones of H37Rv origin (panel B) exhibited distinct strong signals indicating the immunoreactivity of these clones (FIG. 1). All these clones were also tested for immunoreactivity with NHS. However, with the exception of 3 clones which showed a slight reactivity to NHS, none of the clones reacted with NHS, thereby indicating that these expressed mycobacterial antigens reacted selectively with TB sera. Thus, this procedure resulted in the forthright identification of recombinant clones encoding mycobacterial antigens. This strategy can be generally applicable to mycobacterial gene banks prepared in plasmid or cosmid vectors to identify genes which are expressed in E. coli at least to the limit detectable by the immunoassay.
C. Restriction Mapping of Immunoreactive Mycobacterium bovis BCG DNA Recombinants
The insert DNAs of four of the immunoreactive BCG recombinant DNA clones isolated using the TB sera were mapped with restriction endonucleases. FIG. 2, panel B, shows the genomic DNA restriction site maps deduced for the cloned BCG DNA in four recombinants, in which, A represents Sal I, B, BamH I, E, EcoR I, G, Bgl II, K, Kpn I, P, Pvu I, S, Sac I, X, Xho I. These restriction site maps were then compared with those constructed previously for the five immunodominant antigens of M. tuberculosis/M. bovis BCG (Young, et al., Proc. Natl. Acad. Sci., U.S.A., 82:2583-2587 (1985); Husson, et al., Proc. Natl. Acad. Sci., 84:1679-1683 (1987); Shinnick, et al., Infect. Immun., 55, 7:1718-1721 (1987)) (FIG. 2, panel A). Since the restriction site maps shown in panels A and B have been drawn to the same scale, the differences between the two are apparent. There are no regions of similarity between the restriction site maps of immunoreactive BCG recombinant clones and those of the previously characterized immunodominant antigens of M. tuberculosis/M. bovis BCG. Therefore, one can conclude that the cloned BCG DNA inserts in the four recombinants are novel.
EXAMPLE II
Isolation and Characterization of a Gene Encoding a BCG Ion-motive ATPase
A. Identification of a Novel BCG Antigen
One of the four immunoreactive BCG clones, pMBB51A, revealed the presence of a protein of Mr 90 kD, on Western blot analysis using TB sera as well as anti-H37Rv polyclonal antiserum raised in rabbits (FIG. 3). Similar Western blot analysis of pMBB51A with a pool of a few anti-mycobacterial monoclonal antibodies (TB 23, TB 71, TB 72, TB 68, TB 78; Engers et al., Infec. Immun., 48:603-605 (1985)) or with normal human sera did not reveal this immunoreactive protein of 90 kD. This confirms that pMBB51A encodes a BCG antigen which is different from those identified previously in BCG, thereby making it a novel antigen.
B. Determination of the Nucleotide Sequence of pMBB51A
In order to further characterize this novel BCG antigen, pMBB51A DNA insert was subjected to nucleotide sequencing. The BamH I-BamH I insert carried in pMBB51A was mapped for additional restriction enzyme cleavage sites. It was determined that there were at a minimum a single Pst I site and 3 Sal I sites in this sequence. Overlapping fragments derived from single and double digests of Sal I, BamH I and Sal I, BamH I and Pst I, and Pst I and Sal I, were subcloned into M13mpl8 and M13mpl9 vectors, in preparation for DNA sequence analysis. DNA sequencing was then carried out using commercially available kits such as the Sequenase system and the T7 system from Pharmacia. Oligonulceotides derived from the determined sequence were synthesized and used as primers to complete the sequence of the larger inserts. Several areas of compression were encountered during the sequencing and these were resolved by using dITP in the sequencing reactions, and by changing the reaction conditions. The complete nucleotide sequence of the pMBB51A insert DNA was determined by sequencing both the strands using dGTP as well as dITP. The DNA sequence of the pMBB51A insert was determined to be 3.25 kb long with a GC content of 67.1% and is shown in FIG. 4.
The determination of the DNA sequence of the 3.25 kb insert of clone pMBB51A (FIG. 4) permitted the elucidation of the amino acid sequence of the 90 kD BCG antigen. In FIG. 4, nucleotides are numbered from the left end of the pMBB51A insert DNA.
A search of pMBB51A insert DNA sequence for possible ORFs in all three reading frames revealed the longest ORF of 2286 bp encoding a polypeptide of 761 amino acids on one of the strands. The other strand was found to have a smaller URF of 1047 bp capable of encoding a polypeptide of 349 amino acids. The longest ORF encoding a 761 amino acid long protein corresponded to a deduced molecular weight of 79 kD which came closest to the immunoreactive BCG protein with apparent molecular weight of 90 kD, seen on the Western blot. The deduced amino acid sequence for this protein is given below the nucleotide sequence in FIG. 4.
The location of this ORF on the pMBB51A insert DNA was such that there were long stretches of flanking DNA sequences, devoid of any meaningful ORFs, present on either side. This precluded the expression of this ORF from the pBR322 Tet gene promoter and instead suggested that this ORF was being expressed from its own promoter in pMBB51A. This also suggested that E. coli may correctly utilize the M. bovis BCG transcription and translation start and stop sites in this gene.
Immediately upstream of the ORF, regulatory sequences closely matching the −35, −10 and Shine-Dalgarnó sequences of E. coli, (Rosenberg, et al., Annul. Rev. Genet., 13:319-353 (1979)) were identified. The spacing between these three regulatory motifs was also very well conserved. Although the other mycobacterial promoters sequenced (Dale, et al., Molecular Biology of the Mycobacteria, chap. 8, 173-198 (1990)) show some differences from the E. coli consensus sequences in all the three regions −35, −10 and SD, the regulatory elements of pMBB51A DNA showed a maximum degree of sequence identity with E. coli in the −35 and SD sequence elements with a single mismatch in each element, and about 50% sequence identity in the Pribnow box. All the above features clearly indicated that this region is the promoter region for the mycobacterial gene contained in pMBB51A. The extent of similarity between this BCG promoter sequence and a typical E. coli promoter is remarkable and explains the functional activity of this promoter, unlike many other mycobacterial promoters, in E. coli. The translation initiation codon in this ORF was ATG at position 508 while a single translation termination codon TGA was identified at position 2790. Potential transcription termination structures capable of forming stem and loop conformations were identified in the region 3′ to this ORF. The pMBB51A ORF thus represented a monocistronic gene rather than an operon. The promoter region of MBB51A gene is capable of directing gene expression in E. coli as well as in mycobacteria. This promoter sequence is useful for directing expression of mycobacterial genes in E. coli. Further, this promoter sequence can also be used to express homologous and/or heterologous genes in a mycobacterium, thus providing a key element for the development of gene expression systems in mycobacteria.
In order to derive information about the possible biological function of the MBB51A protein, the amino acid sequence of this protein was used to search for homology against available sequences in the PIR Protein Database Release 20 (Table I) and a Genebank Nucleic Acid Database (Table II) using the Fast A suite of programmes written by (Lipman and Pearson, Proc. Natl. Acad. Sci., USA, 85:2 (1988)). The MBB51A protein sequence exhibited homology to a family of ion-motive ATPases from different organisms, ranging from bacteria to mammals. The 13 best scores from a search with ktuple 2 are shown in the upper panel of Table I and 10 best scores from a search with ktuple 1 are shown in the lower panel. In each case, MBB51A protein exhibited maximum homology (75.9% homology in a 593 amino acid overlap with 31.9% identity to a K+ transporting ATPase of S. faecalis (Solioz et al., 1987). The next best homology was observed with the B-chain of K+ transporting ATPase of E. coli (Hesse, et al., Proc. Natl. Acad. Sci., U.S.A., 81:4746-4750 (1984)) (68.8% homology in a 397 amino acid overlap with 24.2% identity). A lesser extent of homology was also seen with H+, Ca++ and Na+−ATPases from different organisms. The results of homology search thus indicated that MBB51A protein is an ion-motive ATPase of M. bovis BCG and is closely related to the other bacterial ion-motive ATPases. This is the first report of the cloning and identification of such an ATPase in mycobacteria. The BCG ion-motive ATPase showed homologies with other ion-motive ATPases with overlapping regions ranging in size from 593 amino acids in case of S. faecalis to 82 amino acids as in case of L. donovani, (Meade, et al., Mol. Cell Biol., 7, 3937-3946 (1987)), though most of the regions of sequence identity or conservation were localized in the C-terminal half of the MBB51A protein. Further, a region of 30 amino acids in the C-terminal half of MBB51A protein was found to be shared with most of these ATPases, thereby suggesting the functional importance of this region. Detailed alignment of MBB51A protein with the K+ ATPases of S. faecalis and E. coli also indicated that several residues were conserved between the three ATPases, including the ones that are invariant in all ATPases from bacteria to man.







TABLE I







RESULTS OF HOMOLOGY SEARCH OF MBB51A


AMINO ACID SEQUENCE AGAINST PIR PROTEIN DATABASE










LOCUS
SHORT DEFINITION
initn
opt













ktuple: 2





>A29576
Potassium - transporting ATPase Strepto-
547
792



coccus


>PWECBK
Potassium - transporting ATPase, β chain -
314
270



E. coli


>A25939
Proton - transporting ATPase - Neurospora
168
186


>A25823
Proton - transporting ATPase - Yeast
166
184


>PWRBFC
Calcium - transporting ATPase, fast twitch
152
158



skele


>PWRBSC
Calcium - transporting ATPase, slow twitch
135
157



skele


>A25344
Potassium - transporting ATPase - Rat
78
155


>RDEBHA
Mercuric reductase -Shigella flexneri
99
142



plasmid


>RDPSHA
Mercuric reductase (transposon Tn501)
74
124


>RGPSHA
Mercuric resistance operon regulatory p
79
109


>A24639
Sodium/potassium-transporting ATPase,
92
82



alpha


>A24414
Sodium/potassium-transporting ATPase,
92
82



alpha


>B24862
Sodium/potassium-transporting ATPase, beta
83
82









The PJR protein data base (2378611 residues in 9124 sequences) was scanned with the FASTA program. The mean of the original initial score was 27.2 with a standard deviation of 6.9. Initial scores (initn) higher than 75.6 are 6 standard deviations above the average, a level of significance that usually indicates biological relatedness. Optimization (opt) generally will improve the initial score of related proteins by introducing gaps in the sequence. Unrelated sequences usually do not have their scores improved by optimization.
















ktuple: 1





>A29576
potassium-transporting ATPase - Strepto-
744
792



coccus


>PWECBK
potassium-transporting ATPase, β chain -
386
270



Esche


>A25939
Proton -transporting ATPase - Neurospora
310
186



crassa


>A25823
proton-transporting ATPase -Yeast (Sacchar-
317
184



omy)


>B24639
Sodium/potassium-transporting ATPase, al-
158
163



pha (+


>A24639
Sodium/potassium-transporting ATPase, al-
175
160



pha ch


>C24639
Sodium/potassium-transporting ATPase, al-
192
159



pha (II


>PWRBFC
Calcium-transporting ATPase, fast twitch
240
158



skele


>PWSHNA
Sodium/potassium-transporting ATPase, al-
214
158



pha skele


>A24414
Sodium/potassium-transporting ATPase, al-
214
158



pha chain
















TABLE II







RESULTS OF HOMOLOGY SEARCH OF MBB51A AMINO ACID


SEQUENCE AGAINST GENBANK NUCLEIC ACID SEQUENCE


DATABASE










LOCUS
SHORT DEFINITION
initn
opt





ktuple: 2





>STRATPK
S. faecalis K+ ATPase, complete cds.
537
800


>ECOKDPABC
E. coli kdpABC operon coding for Kdp-
314
270



ATpase


>YSPPMA1A
S. pombe H+ ATPase, complete cds.
135
188


>NEUATPASE
N. crassa plasma membrane ATPase,
133
186



complete


>NEUATPPM
Neurospora crassa plasma membrane
131
186



H+ ATPase


>YSCPMA1
Yeast PMA1 for plasma membrane
166
184



ATPase


>M17889
FIG. 2 N of L. donovani ATPase and
166
170


>M12898
Rabbit fast twitch skeletal muscle Ca++
140
158



ATPas


>RABATPAC
Rabbit Ca + Mg dependent Ca++
142
157



ATPase mRNA, co


>NR1MER
Plasmid NR1 mercury resistance (mer)
100
143



operon.







ktuple: 1










>STRATPK
S. faecalis K+ ATPase gene,
744
800



complete cds.


>SYNCATPSB
Cyanobacterium Synechococcus 6301
379
422



DNA for AT


>ECOKDPABC
E. coli kdpABC operon coding for Kdp-
379
270



ATPase p


>YSPPMA1A
S. pombe H+ ATPase gene, com-
275
188



plete cds.


>NEUATPASE
N. crassa plasma membrane ATPase
311
186



gene, comple


>NEUATPPM
Neurospora crassa plasma membrane
302
186



H+ ATPase


>YSCPMA1
Yeast PMA1 gene for plasma mem-
317
184



brane ATPase


>JO4004
Leishmania donovani. cation trans-
322
170



porting ATP


>M17889
FIG. 2 Nucleotide seguence of L.
306
170



donovani


>RATAPA2
Rat Na+, K+ ATPase alpha (+) isoform
158
163



catalytic









The KdpB protein of E. coli and possibly the S. faecalis K+ ATPase are members of E1E2-ATPases which are known to form an aspartyl phosphate intermediate, with cyclic transformation of the enzyme between phosphorylated and dephosphorylated species. By analogy to other ATPases, the phosphorylated Asp residue (D) (Furst, et al., J. Biol. Chem., 260:50-52 (1985)) was identified at position 443 in the MBB51A ATPase. This residue is the first of a pentapeptide sequence DKTGT that has been conserved in ATPases from bacteria to man, and must form an essential element of the catalytic site. Similarly, proline (P) at position 400 in MBB51A ATPase was found to be an invariant amino acid in other ATPases and is predicted to be located in a membrane spanning domain. Such membrane buried proline residues have been hypothesized to be required for the reversible conformational changes necessary for the regulation of a transport channel (Brandl, et al., Proc. Natl. Acad. Sci., U.S.A., 83:917-921 (1986)). In addition, other sequence motifs believed to be functionally important in other ion-motive ATPases were also found to be conserved in the MBB51A ATPase. These include a Gly (G) (Farley and Faller, J. Biol. Chem., 260:3899-3901 (1985)) at position 521 and Ala (A) (Ohta, et al., Proc. Natl. Acad. Sci., U.S.A., 83:2071-2075 (1986)) at position 646, and are shown in FIG. 5.
Since the MBB51A ATPase was homologous to membrane associated ATPases, characterization of the membrane associated helices in MBB51A protein was performed by computer algorithms. Using a hydropathy profile (Rao, et al., Biochem. Biophys. Acta., 869:197-214 (1986)), seven transmembrane domains in the MBB51A protein were identified and are shown in Table III and FIG. 5. Nearly the same transmembrane domains were also identified using the hydrophobic moment plot (Eisenberg et al., J. Mol. Biol., 179:125-142 (1984)) and are also shown in Table III and FIG. 5. The average size of a transmembrane domain is around 21 residues, because 21 residues coil into an α-helix approximately the thickness of the apolar position of a lipid bilayer (32 Å). This size of a transmembrane domain is, however, flexible within the range of a few amino acids, as determined by the functional properties of a given membrane-associated protein. The transmembrane domains identified in MBB51A protein, range in size from 20-37 residues. The first six transmembrane domains span the membrane only once, as indicated by both the hydropathy profile and the hydrophobic moment plot. The seventh transmembrane domain may traverse the membrane twice. These features along with the membrane buried proline (P) at position 400, are in accordance with the channel transport functions of ion-motive ATPases, involving a reversible change in the conformation of these proteins. Such transmembrane domains further define the intracellular and extracellular domains of this molecule. See FIG. 5.









TABLE III





Transmembrane
Eisenberg
Rao & Argos


Domain in FIG. 5
Method
Method







1
102-122
 98-125


2
129-149
127-147


3
164-184
164-185


4
199-219
198-220


5
361-381
360-382


6
387-407
387-419


7
703-723
695-732









The hydropathy profile of MBB51A protein was nearly superimposable over that of S. faecalis K+ ATPase, even though the MBB51A ATPase has at the N-terminus, 154 extra amino acids, which were absent in S. faecalis. This clearly puts in evidence the strong evolutionary conservation of the broad domain structure between these two proteins, making it more likely for the two proteins to have a similar three dimensional structural organization.
Based on the hydropathy profile and secondary structure predictions, a schematic model of the MBB51A ATPase is presented in FIG. 5. This model comprises at least seven transmembrane domains which span the membrane, once are indicated along with the respective amino acid positions in FIG. 5. This model further defines extracellular and intracellular domains of the MBB51A protein. Many of the residues which have been shown to be functionally important in other ion-motive ATPases and are also conserved in the MBB51A protein, are also shown. Of these, proline (P) at position 400 is membrane-buried whereas as aspartic acid(D) at 443, glycine (G) at 521 and alanine (A) at 646, face the cytoplasm.
In order to determine whether the gene encoding MBB51A ion-motive ATPase is present in other mycobacterial strains related or unrelated to BCG, like the virulent strain M. tuberculosis H37Rv and other non-tuberculous, non-pathogenic mycobacteria like M. vaccae and M. smegmatis, Southern blot hybridization with genomic DNA from the above species was performed, using as probe BCG insert DNA from pMBB51A. As shown in FIG. 6, DNA hybridizable with the pMBB51A insert DNA was also present in M. tuberculosis H37Rv DNA but not in M. smegmatis and M. vaccae. This indicated that the M. tuberculosis H37Rv homologue of the pMBB51A gene has a similar genetic organization as seen in M. bovis BCG DNA, and is present on a 3.25 kb BamH I fragment.
The availability of novel Mycobacterium bovis BCG and/or Mycobacterium tuberculosis H37Rv antigens make it possible to address basic biochemical, immunological, diagnostic and therapeutic questions still unanswered about tuberculosis and Mycobacterium tuberculosis. For example, Mycobacterium tuberculosis specific antigenic determinants can be used to develop simple and specific seroepidemiological tests to screen human populations. Such serological tests are highly specific because of the use of antigenic determinants determined by the approaches described above and known to be unique to Mycobacterium tuberculosis H37Rv. Such serological tests are useful for early diagnosis of tuberculosis, thus permitting early treatment and limiting transmission of the disease from infected individuals to others.
Resistance to tuberculosis is provided by cell mediated immunity. The antigens identified here can be further used to determine which segments of these antigens are recognized by Mycobacterium tuberculosis specific T-cells. A mixture of peptides recognized by helper T-cells provides a specific skin test antigen for use in assessing the immunological status of patients and their contacts. A mixture of such peptides is also useful in evaluating rapidly the immunological efficacy of candidate vaccines. In addition peptides recognized by Mycobacterium tuberculosis specific T-cells can be components of a vaccine against the disease.
Knowledge of the complete nucleotide sequence of pMBB51A DNA insert provides a rich source of sequence information which can be used to design appropriate primers for PCR amplification of mycobacterial genomic DNA fragments. The ion-motive ATPase of BCG has areas of heavily conserved sequences (for, e.g., the ATP binding site) which are expected to be the same for all mycobacterial species and areas of sequence divergence (for, e.g., the N-terminal region) which are different in different mycobacterial species. Based on this knowledge primers can be designed either from the conserved regions or from the diverged regions to identify whether in a given sample the target DNA is mycobacterial versus non-mycobacterial, and in case of mycobacterial DNA, which mycobacterial species the DNA belongs.
Such amplification schemes are useful for the development of highly sensitive and specific PCR amplification based diagnostic procedures for mycobacteria. The observation that the 3.25 kb pMBB51A DNA insert is present in Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG and is absent in avirulent Mycobacterium vaccae and Mycobacterium smegmatis, which have bearing on other aspects of the biological differences between these species, manifest in terms of virulence, growth characteristics and metabolism.
Recombinant vaccines can also be constructed by incorporating the DNA encoding all or part of the membrane-associated polypeptides of the invention into an appropriate vaccine vehicle. For example, all or part of the DNA encoding the 79 kD Mycobacterium bovis BCG protein or a portion of the protein can be incorporated into a vaccine vehicle capable of expressing the said DNA. Such a vaccine vehicle could be a virus for, e.g., vaccinia virus, etc., or a bacterium, e.g., mycobacteria, Salmonella, Vibrio, Bacillus, Yersinia, Bordetella, etc. to produce a vaccine capable of conferring long-lasting immunity on individuals to whom it is administered.
A special feature of the 79 kD BCG ion-motive ATPase is that it is a membrane bound antigen. Therefore, it can be used to link foreign DNA sequences encoding antigenic epitopes (B-cell epitopes or T-cell epitopes) of interest, with this gene or a portion of this gene in a manner which causes the foreign epitope to be used as an immunogen. Such linkages can be engineered into extracellular or intracellular domains of MBB51A protein, or into a combination of both types of domains. Engineering of immunogenic foreign epitopes into MBB51A DNA is accomplished by standard recombinant DNA methods known to those skilled in the art. Some of these methods involve use of unique restriction sites, in vitro mutagenesis and/or PCR-related methods. One such convenient method involves the use of a unique NdeI site at position 1090 in the MBB51A DNA where foreign DNA can be inserted. Grafting of epitopes on the cell surface induces rapid antibody response by virtue of the epitope being well-exposed on the bacterial cell, which in turn leads to direct activation of B cells. In addition, intracellular localization of an epitope induces B cell memory and a proficient T cell response. Examples of epitopes of interest known to be involved in the immune response to various pathogens include epitopes from E. coli LT toxin, foot and mouth disease virus, HIV, cholera toxin, etc.
Thus, the 79 kD antigen is useful in the design of recombinant vaccines against different pathogens. Such vaccines comprise a recombinant vaccine vehicle capable of expressing all or part of the 79 kD membrane-associated protein of mycobacteria, into which foreign epitopes have been engineered, such that the foreign epitopes are expressed on the outer surface and/or on the inner side of the cell membrane, thereby rendering the foreign epitopes immunogenic. The vaccine vehicle for this purpose may be a cultivable mycobacterium for, e.g., BCG. In these applications, the BCG ion-motive ATPase gene can be borne on a mycobacterial shuttle vector or alternately the foreign DNA encoding antigenic epitopes of the immunogenic polypeptides can be inserted into the mycobacterial genome via homologous recombination in the ion-motive ATPase gene or random integration. Such a process yields stable recombinant mycobacterial strains capable of expressing on their surface and/or in the cytoplasm antigenic sequences of interest, which can, for example, provide protection against a variety of infectious pathogens. Targeting of recombinant antigens to the cell-wall is attractive not only because of the high immunogenicity of mycobacterial cell-walls but, in addition, because of concerns with the introduction of a live vaccine in populations with a high prevalence of HIV seropositivity. Additionally, based on the MBB51A protein, a non-living but immunogenic recombinant cell surface subunit vaccine can also be developed to provide a useful alternative to live vaccines. Alternately, other bacterial, viral or protozoan vaccine vehicles could be transformed to generate such recombinant vaccines. Examples of potential vaccine vehicles include vaccinia virus, pox-viruses, Salmonella, Yerisinia, Vibrio, Bordetella, Bacillus, etc.
Further, using such an approach, multivalent recombinant vaccines which allow simultaneous expression of multiple protective epitopes/antigens of different pathogens, could also be designed.
Equivalents
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific materials and components described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

## Claims

1. A method of detecting the presence of antibodies to virulent Mycobacterium in a biological sample, said method comprising:
combining said sample with a polypeptide having the amino acid sequence of SEQ ID NO:2, or an antigenic determinant thereof; and
detecting antibodies bound to said polypeptide; wherein said Mycobacterium is M. bovis, M. tuberculosis, M. leprae, M. africanum, M. microti, M. avium, M. intracellulare or M. scrofulaceum.

2. The method of claim 1, wherein said polypeptide is immobilized on a solid support.

3. The method of claim 2, wherein said solid support is nitrocellulose.

4. The method of claim 1, wherein said sample comprises one or more of sputum, blood, and serum.

5. The method of claim 1, wherein said detecting is by a qualitative detection system.

6. The method of claim 5, wherein said qualitative detection system is a horseradish peroxidase-protein A detection system.

7. The method of claim 1, wherein said detecting is by a quantitative detection system.

8. The method of claim 7, wherein said qualitative detection system is a radioimmunoassay.

9. The method of claim 1, further comprising:
combining a control biological sample with said polypeptide; and comparing the detection of said binding to the binding of antibodies in the control sample with said polypeptide.

10. A method of detecting the presence of Mycobacterium in a biological sample, said method comprising:
Lysing the cells in said sample;
Combining said lysate with antibodies to a polypeptide having the amino acid sequence of SEQ ID NO:2 or an antigenic determinant thereof; and
detecting said antibodies bound to polypeptide in said lysate;
wherein said Mycobacterium is M. bovis, M. tuberculosis, M. leprae, M. africanum, M. microti, M. avium, M. intracellulare or M. scrofulaceum.

11. The method of claim 10, wherein said Mycobacterium is M. bovis.

12. The method of claim 10, wherein said lysate is immobilized on a solid support.

13. The method of claim 12, wherein said solid support is nitrocellulose.

14. The method of claim 10, wherein said detecting is by a qualitative detection system.

15. The method of claim 14, wherein said qualitative detection system is a horseradish peroxidase-protein A detection system.

16. The method of claim 10, wherein said detecting is by a quantitative detection system.

17. The method of claim 16, wherein said quantitative detection system is a radioimmunoassay.

18. The method of claim 10, further comprising:
culturing a diagnostic sample to produce colonies of bacteria present therein, whereby said culture represents said biological sample.

19. A method of detecting the presence of antibodies to a virulent Mycobacterium in a biological sample, said method comprising: combining said sample with a purified polypeptide of a Mycobacterium other than M. bovis BCG,
wherein said polypeptide is a homolog of the protein of SEQ ID NO:2; is an immunogenic membrane-associated protein of said Mycobacterium, and is encoded by a DNA which is hybridizes with a DNA probe having the complete sequence represented in SEQ ID NO:1 under conditions where, on a Southern blot said probe will identify single 3.25 kb BamHI fragments from M. bovis BCG and M tuberculosis H37Rv DNA, but will not hybridize with BamHI-digested DNA from either M. smegmatis or M. vaccae; and detecting an antibody bound to said polypeptide that is homologous to SEQ ID NO:2 and that is obtained from a virulent strain of Mycobacterium.

20. The method of claim 10, wherein said Mycobacterium is M. tuberculosis.