EP0345299A1 - Mycobacterium tuberculosis genes encoding protein antigens - Google Patents

Abstract

Mycobacterium tuberculosis genes encoding five immunologically active proteins were isolated by systematically screening a lambda gt11 recombinant DNA expression library with a collection of murine monoclonal antibodies directed against protein antigens of this pathogen. One of the M. tuberculosis antigens, a 65kD protein, has determinants common to M. tuberculosis and M. leprae. Additionally, protein-coding genes from other mycobacteria M. africanum, M. smegmatis, M. bovis BCG and M. avium were isolated. The isolation and characterization of genes encoding major protein antigens of M. tuberculosis allows the development of reagents useful for the diagnosis, prevention and treatment of tuberculosis. They can be used, for example, in the development of epidermal tests, serodiagnosis tests and vaccines against tuberculosis.

Description

MYCOBACTERIUM TUBERCULOSIS GENES AND ENCODING PROTEIN ANTIGENS

Description

Background Tuberculosis was the major cause of infectious mortality in Europe and the United States in the 19th and early 20th centuries. Dubos, R. and J. Dubos, The White Plague: Tuberculosis, Man and Society, Little Brown & Co., Boston, MA, (1952). Today, it remains a significant global health problem.

For example, in the United States there are over 20,000 new cases of tuberculosis diagnosed annually. In addition, the steadily declining incidence of tuberculosis evident in preceding years appears to have changed course, reaching a plateau in 1985 and showing an increase in the first half of 1986. Centers for Disease Control, Morbidity/Mortality, Weekly Report, 34 : 774 (1986); and Centers for Disease Control, Morbidity/Mortality, Weekly Report, 35:774 (1986).

Worldwide, tuberculosis remains widespread and constitutes a health problem of major proportions, particularly in developing countries. The World Health Organization estimates that there are ten million new cases of active tuberculosis per year and an annual mortality of approximately three million. Joint International Union Against Tuberculosis and World Health Organization Study Group, Tubercle, 63:157-169 (1982).

Tuberculosis is caused by Mycobacterium (M.) tuberculosis or Mycobacterium (M.) bovis, which are the 'tubercle bacilli' of the family Mycobacteriaceae. M. bovis is a species which causes tuberculosis in cattle and is transmissible to humans and other animals, in whom it causes tuberculosis. At present, nearly all tuberculosis is caused by respiratory infection with M. tuberculosis. Infection may be asymptomatic in some, but in other individuals, it produces pulmonary lesions which lead to severe debilitation or death. Resistance to tuberculosis is provided by cell-mediated immune mechanisms.

Mycobacteria are aerobic, acid-fast, non-spore- forming, non-motile bacili with high lipid contents and slow generation times. M. leprae is the etiologic agent of leprosy and, among the other mycobacteria, the only major pathogen. Bloom, B.R. and T. Godal, Review of Infectious Diseases, 5:765-780 (1983). However, other mycobacterial species are capable of causing disease. Wallace, R.J. et.al., Review of Infectious Diseases, 5:657-679 (1984). M.avium, for example, causes tuberculosis in fowl and in other birds. Members of the M. Avium-intracellularae complex have become important pathogens among individuals with acguired immunodeficiency syndrome (AIDS). Certain groups of individuals with AIDS have a markedly increased incidence of tuberculosis as well. Pitchenik, A.E. et. al., Annals of Internal Medicine, 101:641-645 (1984). Diagnostic and immunoprophylatic measures for mycobacterial diseases have changed little in the past half century. Tuberculin, developed by Koch as a cure for tuberculosis in the late 1800s, is an M. tuberculosis filtrate of complex and poorly-defined composition. It is used as a skin test antigen to detect prior exposure to the bacillus. Enrichment of the protein fraction of this material in the 1930's produced the purified protein derivative (PPD) which is still used to diagnose exposure to tuberculosis. Seibert, F.M. et. al., American Review of Tuberculosis, 30(Suppl.):705-778 (1934). Its usefulness is limited, however, by its lack of specificity and its inability to distinguish active disease from prior sensitization by contact with M. tuberculosis or cross-sensitization to other mycobacteria. Young, R.A. and R.W. Davis, Proceedings of the National Academy of Sciences, USA, 80:194-1198 (1983).

Bacille Calmette Guerin (BCG), an avirulent strain of M. bovis, has been used widely as a live vaccine against tuberculosis for over 50 years. Calmette, A., C. et.al., Bulletin of the Academy of Medicine Paris, 91 : 181-196 (1924). During that time, numerous studies have shown that BCG has protective efficacy against tuberculosis. These studies are reviewed by F. Luelmo in American Review of Respiratory Diseases, 125 (pt. 2):70-72 (1982). However, more recently, a major trial of BCG in India indicated that such a vaccine was not protective against tuberculosis in this setting. World Health Organization WHO Technical Report Series, 651 (1980). Presently available approaches to diagnosing, preventing and treating tuberculosis are limited in their effectiveness and must be improved if a solution is to be found for the important public health problem tuberculosis represents worldwide.

Summary of the Invention

The present invention is based on the isolation of genes encoding immunogenic protein antigens of the tubercle bacillus Mycobacterium tuberculosis (M. tuberculosis). Genes encoding such protein antigens have been isolated from a recombinant DNA expression library of M. tuberculosis DNA. Genes encoding proteins of four additional mycobacteria have also been isolated and restriction maps produced. in particular, genes encoding five immunodominant protein antigens of the tuberculosis bacillus (i.e., those M. tuberculosis proteins of molecular weight 12,000 daltons (l2kD), 14kD, 19kD, 65kD and 71kD have been isolated by probing a lambda gtll expression library of M. tuberculosis DNA with monoclonal antibodies directed against M. tuberculosis-specific antigens.

Recombinant DNA clones producing the specific antigenic determinants recognized by the monoclonal antigens were also isolated in this manner. DNA from such recombinant lambda gtll clones was mapped with restriction endonucleases; the restriction maps for genes encoding the five immunodominant protein antigens (i.e., genes encoding the 12kD, 14kD, 19kD, 65kD and 71kD proteins) were deduced. The nucleotide sequence of three of the genes have been determined and, in each case, the amino acid sequence of the encoded protein has been deduced.

Brief Description of the Drawings Figure 1 shows restriction maps of M. tuberculosis DNA. Recombinant DNA clones isolated with monoclonal antibodies directed against the 12kD, 14kD, 19kD, 65kD and 71kD protein antigens were mapped with restriction endonucleases. The insert DNA endpoints are designated left (L) or right (R) in relation to lac Z transcripts which traverse the insert from right to left. Restriction sites are represented as follows: A, Sal I; B, BamHI; E, EcoRI; G, BglII; K, KpnI; P, PvuI; S, Sad; X, XhoI. Figure 2 shows arrays of antigens from M. tuberculosis recombinant DNA clones probed with rabbit hyperimmune serum. The code of the recombinant DNA clones shown on the numbers filter is: 1, Y3275; 2, Y3274; 3, Y3279; 4, Y3277; 5, Y3247; 6, Y3272; 7, Y3150; 8, Y3254; 9, Y3147; 10, Y3163; 11, Y3179; 12, Y3191; 13, Y3252; 14, Y3178; 15, Y3180; 16, Y3143; 17, lambda gtll. Clones 1, 5, 6, 7, 9 and 16 are M. tuberculosis recombinants described in the following section. Clones 10, 11, 14 and 15 are M. leprae recombinants expressing epitopes of the 18kD, 28kD, 36kD and 65kD antigens, respectively. Clones 2, 3, 4, 8, 12, 13 are uncharacterized recombinants from the lambda gtll M. tuberculosis and M. leprae libraries. Clone 17 is a nonrecombinant lambda gtll control.

Figure 3 shows arrays of recombinant mycobacterial antigens probed with monoclonal antibodies to assess the extent of cross-reactivity between recombinant protein antigen of M. tuberculosis and of M. leprae. The array of clones is identical to that shown in Figure 2. Antibody probes and the antigen sizes recognized are: 1, IT-11 (71kD); 2, IT-31 (65kD); 3, IT-16 (19kD); 4, IT-1 (14kD); 5, IT-3 (12kD). Figure 4 shows restriction maps of DNA encoding four proteins (71kD, 65kD, 19kD and 14kD) of M. tuberculosis and four proteins (71kD, 65kD, 19kD and 14kD) of M. bovis BCG. Restriction sites are represented as follows: A, AatII; B, BamHl; C, BelI; D, DraIII; E=EcoRI; G, BglII; H, HinfI; K, KpnI; P, PstI; S, SalI; V, PvuI and X, XhoI.

Figure 5 is a comparison of restriction maps of the gene encoding the 65kD protein of 6 mycobacteria (M. leprae, M. tuberculosis, M. africanum, M. bovis BCG, M. smegmatis, M. avium). Restriction sites are as follows: B, BamHl; K, KpnI; N, SacI; P, PvuI; S,

SalI; X, XhoI.

Figure 6 is the nucleotide sequence of the region containing the M. tuberculosis 19kD gene. The deduced amino acid sequence of the encoded protein is also represented (protein start position, nucleotide 1110; protein stop position, nucleotide

1586).

Figure 7 is the nucleotide sequence of the region containing the M. tuberculosis 71kD gene and the deduced amino acid sequence of the encoded protein.

Figure 8 is the nucleotide sequence of the region containing the M. tuberculosis 65kD gene. The deduced amino acid sequences of the two long open reading frames are presented in one letter code over (540) or under (517) the appropriate triplets.

Detailed Description of the Invention

The invention described herein is based on the isolation of genes encoding immunogenic protein antigens of the bacillus M. tuberculosis, which is the major etiologic agent of tuberculosis. In particular, it is based on the isolation, using monoclonal antibodies directed against M. tuberculosis-specific antigens, of genes encoding five immunogenic protein antigens of the tuberculosis bacillus; these five antigens are immunodominant. Immunogenic antigens are those which elicit a response from the immune system. Immunodominant protein antigens are immunogenic antigens against which the immune system directs a significant portion of its response. Genes encoding M. tuberculosis antigens of molecular weight 12,000 daltons (12kD), 14kD, 19kD, 65kD and 71kD were isolated in this manner.

Isolation and characterization of major protein antigens of M. tuberculosis, as described herein, make it possible to develop more effective tools for the prevention, diagnosis, and treatment of tuberculosis. Identification and isolation of genes encoding five immunodominant M. tuberculosis protein antigens, as well as of the five protein antigens, are described below; uses of the genes and encoded products are also described. M. bovis BCG DNA clones were also isolated for the genes encoding the 71kD, 65kD, 19kD and 14kD proteins. In order to compare M. bovis BCG and M. tuberculosis genes encoding proteins of similar molecular weight, restriction endonuclease maps were determined for DNA segments containing each of the genes. Restriction maps for each of these genes is represented in Figure 4.

In addition, DNA clones were isolated for the genes encoding the 65kD protein from M. africanum, M. smegmatis and M. avium. Restriction endonuclease maps were determined for DNA segments containing each of these genes. The restriction maps for these genes, as well as for the genes encoding the 65kD protein of M. tuberculosis, M. bovis BCG and M. leprae, are represented in Figure 5. I. Construction of a recombinant expression library of M. tuberculosis DNA

A recombinant DNA expression library of M. tuberculosis DNA was constructed using lambda gtll. The library was constructed with M. tuberculosis genomic DNA fragments in such a way that all protein-coding sequences would be represented and expressed. Young, R.A., B.R. Bloom, C.M. Grosskinsky, J. Ivanyi, D. Thomas and R.W. Davis, Proceedings of the National Academy of Sciences, USA, 82:2583-2587 (1985).

Lambda gtll is a bacteriophage vector which is capable of driving the expression of foreign insert DNA with E. coli transcription and translation signals. Lambda gtll expresses the insert DNA as a fusion protein connected to the E. coli Beta-galactosidase polypeptide. This approach ensures that the foreign DNA sequence will be efficiently transcribed and translated in E. coli. This approach is also useful in addressing the problem of the highly unstable nature of most foreign proteins; fusion proteins are often more resistant to proteolytic degradation than is the foreign polypeptide alone. Lambda gtll and the E. coli strain used (Y1090) have been described previously. Young, R.A. et al., Proceedings of the National Academy of Sciences, USA, 80: 1194-1198 (1983); Young, R.A. and R.W. Davis, Science, 222:778-782 (1983). The teachings of these publications are incorporated herein by reference. The library constructed in this manner has a titer of 1× 10¹⁰ pfu/ml. and contains approximately 40% recombinants with an average insert size of 4kB.

II. Screening of the lambda gtll M. tuberculosis library with antibody probes Murine monoclonal antibodies to protein antigens of M. tuberculosis were used individually to probe the M. tuberculosis recombinant DNA library. This work is described below and with specific reference to the 65kD antigen in the Exemplification. The antibodies used as probes and the sizes of the antigens to which they bind are shown below.

M. tuberculosis

Antibody Antigen

IT-3 12kD

IT-20 14kD

IT-19 19kD

IT-27 19kD

IT-17 23kD

IT-29 23kD

IT-15 38kD

IT-21 38kD

IT-23 38kD

IT-13 65kD

IT-31 65kD

IT-33 65kD

IT-11 71kD

Engers, H.D. et al., Infectious Immunology

51:718-720 (1986). All monoclonal antibodies were used at approximately 1:200 to 1:300 dilution in 50mM Tris-HCl pH8/150 mM NaCl/.05% Tween 20.

Screening of the lambda gtll recombinant DNA library was performed as described by Young et al. in Proceedings of the National Academy of Sciences, USA, 82:2283-2587 (1985), the teachings of which are incorporated herein by reference. One modification was made in the method described by Young and co-workers: 1% bovine serum albumin was used in place of 20% fetal calf serum to decrease background.

Briefly, cloned lambda gtll recombinants were arrayed on lawns of E. coli Y1090. The phage were grown, antigen expression was induced and the antigens were blotted and probed with serum. Detection of signal-producing plaques was performed with a biotinylated secondary antibody system (Vectastain, Vector Laboratories, Burlingame, CA) or with an alkaline phosphatase conjugated secondary antibody system (Protoblot, Promega Biotec, Madison, WI), both used according to manufacturer's instructions. Signal-producing clones were isolated using antibodies directed against protein antigens of molecular weight 12kD, 14kD, 19kD and 65kD and 71kD.

In each case, similar numbers of clones were isolated in screens of approximately 10⁵ recombinant plaques. DNA clones encoding the 23kD and 38kD antigens could not be detected with these anti-bodies, possibly because the native epitope is modified or topographically complex, or because the antigen-antibody interaction is too weak to be recognized by current detection methods.

III. Probing of Arrays of lambda gtll DNA Clones with Antibody Probes 0.2 ml of a saturated culture of Y1090 was added to 2.5 ml of molten LB soft agar, poured onto 100 mm plates containing 1.5% LB agar and allowed to harden at room temperature for 10 min. 100 ul of phage plate stock containing approximately 10¹¹ pfu/ml of the lambda gtll DNA clones of interest were placed into alternate wells of 96-well tissue culture plates. A multi-pronged transfer device was placed briefly in the wells containing phage and then touched lightly to the surface of the plate onto which the soft agar had been poured. The plates were then incubated at 42°C for approximately 3 hours, at which point clear plaques approximately 5mm in diameter were visible. The plates were then overlayed with nitrocellulose filters saturated with 10mM isopropylthiogalactoside (IPTG) and incubated at 37ºC for 3.5 hours. Subsequent processing of filters for detection of antigen was identical to the procedures described for screening of lambda gtll library with antibody probes. Immunoscreening of the lambda gtll library to isolate clones reactive with monoclonal antibodies specific for the 65kD antigen is described in the Exemplification. IV. Recombinant DNA Manipulation DNA from recombinant lambda gtll clones was isolated and mapped with restriction endonucleases by standard techniques. Davis, R.W. et al., Advanced Bacterial Genetics: A Manual for Genetic Engineering, Cold Spring Harbor (1980).

Figure 1 shows the genomic DNA restriction map deduced for each of the genes encoding the five M. tuberculosis antigens and illustrates how each of the cloned DNAs aligns with that map. All clones isolated with monoclonal antibodies directed against any single antigen align with a single genomic DNA segment. This indicates that all clones were isolated because they express the protein of interest rather than an unrelated polypeptide containing a similar or identical epitope. In addition, this result suggests that each antigen is the product of a single gene.

The orientation of each DNA insert in the recombinant clones was determined by restriction analysis . Only among the clones for the 65kD antigen were the inserts found in both possible orientations relative to the direction of lac Z transcription in lambda gtll. This suggests that this protein can be expressed in E. coli from signals independent of those provided by lac Z. Similar results have been obtained for recombinant DNA clones encoding the 65kD antigens of M. bovis and M. leprae. Thole, J.E.R. et al., Infectious Immunology, 50 : 800-806 ( 1985 ) ; Young , R.A. et al . ,

Nature , 3 16 : 450-452 ( 1985 ) . The nucleotide sequences of three regions of the M. tuberculosis DNA were determined: 1) the region containing the M. tuberculosis 19kD gene; 2) the region containing the M. tuberculosis 71kD gene; and 3) the region containing the 65kD gene. The three sequences are represented in Figures 6-8. Sequences were determined using standard techniques, which are described in the Exemplification.

V. Filter hybridization of Insert DNA Arrays of lambda gtll clones were created as described above and incubated at 42° for 5 hours. The plates were then overlayed with nitrocellulose filters and placed at 4°C for 1 hour. Probe DNA was labelled with ³²P by nick translation. Filter hybridization was performed as described by Davis et al. in Advanced Bacterial Genetics: A Manual for Genetic Engineering, Cold Spring Harbor (1980), the teachings of which are incorporated herein by reference. Hybridization conditions were as follows: 50% v/v formamide, 5x SSPE (1x SSPE is 0.18M NaCl, 10mM Na_{1. 5}H_1.5PO₄, lmM Na₂ EDTA, pH 7.0), 1x Denhardt's solution (0.02% w/v Ficoll, 0.02% w/v polyvinyl- pyrrolidone, 0.02% w/v bovine serum albumin), 0.3% NaDodSO₄ at 42°C for approximately 16 hours, followed by washing in 2x SSPE, 0.2% NaDodSO₄ at 45°C.

VI. Recombinant Antigens Recognized by Rabbit Serum

The response of a second animal to an antigen preparation of M. tuberculosis was assessed by examining the reactivity of rabbit anti-M. tuberculosis hyperimmune sera with recombinant antigens. Cloned lambda gtll recombinants were arrayed on lawns of E. coli and probed with the rabbit hyperimmune serum. Anti-M. tuberculosis hyperimmune serum, produced by repeated immunization of rabbits with M. tuberculosis H37Rv culture filtrate, was provided by J. Bennedsen (Statens Seruminstitut, Copenhagen, Denmark). These sera were used at 1:100 dilution.

These sera produced positive signals with lambda gtll clones encoding each of the five M. tuberculosis epitopes which had been isolated with murine monoclonal antibodies (Figure 2). Particularly strong signals were observed with the 65kD and 71kD antigens (Figure 2). These results demonstrate that mice and rabbits can mount an antibody response to the same protein antigens of M. tuberculosis. Clones for the five M. tuberculosis antigens were detected at similar frequencies in the lambda gtll recombinant DNA library. Thus, the number and type of antigen-producing clones isolated with polyclonal serum antibodies should reflect the relative titer and deversity of the individual antibodies in this serum.

To determine whether any of the 5 M. tuberculosis antigens are relatively immunodominant in the rabbit humoral immune response to M. tuberculosis, the M. tuberculosis lambda gtll recombinant DNA library was screened with the rabbit serum. Forty signal-producing clones were isolated, arrayed on lawns of E. coli Y1090 and probed with monoclonal antibodies directed against each of the 5 recombinant M. tuberculosis protein antigens. Remarkably, 17 of the 40 clones (43%) reacted strongly with at least one of the four anti-65kD monoclonal antibodies tested. An additional six clones (15%) reacted strongly with the anti-17kD monoclonal antibody (IT-11). This indicates that a large proportion of the anti-M. tuberculosis antibody present in the rabbit serum was directed against the 65kD antigen of M. tuberculosis and suggests that it is a dominant antigen for the rabbit humoral immune response. Seventeen of the clones did not react with any of the monoclonal antibodies tested, suggesting that the, rabbit sera may identify M. tuberculosis proteins not recognized by the murine antibodies.

VII. Antigenic Relatedness of M. tuberculosis and M. leprae Proteins There is evidence that M. tuberculosis and M. leprae share immunologically important antigens. To assess this further, an investigation of the exact nature of the immunological relatedness among recombinant protein antigens of M. tuberculosis and M. leprae was conducted.

For each of five M. tuberculosis and four M. leprae protein antigens, a single recombinant DNA clone containing most or all of the gene of interest was used to express antigen in the following manner. The recombinant phage clones were arrayed on a lawn of E. coli Y1090, which was then grown and induced for antigen expression.

Antigen immobilized on nitrocellulose filters was then probed with 26 individual anti-M. tuberculosis and M. leprae monoclonal antibodies. Figure 3 shows the array of DNA clones used and the results obtained with the anti-M tuberculosis antibodies IT-1, IT-3, IT-11, IT-16, and IT-31, which recognized proteins of 14kD, 12kD, 71kD, 19kD and 65kD respectively. Table 1 details the full results of these cross-screening experiments, showing the reactivity of antigen expressed from individual recombinant DNA clones with each of the individual monoclonal antibodies. Clones were scored as positive only if the signal produced was clearly greater than the background signal produced by the non-recombinant lambda gtll clone included in/each array.

Several conclusions can be drawn from the results shown in Table 1. Among the 11 monoclonal antibodies that recognize a 65kD antigen, 7 react with the 65kD protein from both mycobacteria (IT-31, C1.1, IIH9 (identical to IT-33), IIC8, T2.3, Y1-2, SA2.D7C), one antibody reacts only with the M. tuberculosis 65kD protein (IT-13), and two antibodies react only with the M. leprae 65kD protein (IIIE9 and IIIC8). One antibody, ML30A, cross-reacts with an antigen in E. coli and could not specifically identify antigen-producing clones. These results indicate that the 65kD protein antigens of M. tuberculosis and M. leprae are homologues and share a number of epitopes. In addition to these shared epitopes, however, both 65kD antigens have epitopes that are specific for one species relative to the other.

No cross-reactivity was observed between other antigens of these two mycobacterial species. Because monoclonal antibodies recognize a single epitope and because only one or a few antibodies were available for each antigen, it is not clear whether the 65kD proteins are the only homologous protein antigens of M. tuberculosis and M. leprae. Among the antigens for which lambda gtll clones have been isolated, the 18kD antigen of M. leprae and the 19kD antigen of M. tuberculosis are of similar size. To determine whether these two antigens are related, the homology of the DNA sequences that encode these antigens was examined. At conditions of moderate stringency, no hybridization was observed between the insert DNA and Y3147 (an M. tuberculosis 19kD clone) and Y3179 (an M. leprae 18kD clone). This indicates no significant homology between the DNA sequences of the insert DNAs of these two clones. This result suggests that the M. tuberculosis 19kD and the M. leprae 18kD proteins are unlikely to be homologous.

As a result of the work described, recombinant DNA clones encoding five major protein antigens of M. tuberculosis were isolated through the use of an extensive collection of well-characterized murine monoclonal antibodies. These five proteins were also found to be major antigens in the rabbit humoral immune response to M. tuberculosis. One of these antigens, the 65kD protein, is shared with another other mycobacterial pathogen M. leprae.

Several lines of evidence indicate that the 65kD antigen is among the most immunodominant of the protein antigens of M. tuberculosis. Eleven of the 25 different M. tuberculosis and M. leprae monoclonal antibodies examined in this study recognized the 65kD recombinant antigen from one or both mycobacteria. In addition, almost half of the recombinant DNA clones isolated with rabbit polyclonal anti-M. tuberculosis sera express the 65kD antigen, reflecting the predominance of antibody to this antigen in these sera.

Considerable evidence indicates that the 65kD antigen plays an important role in the human response to tuberculosis. Antibodies directed against this protein can be detected in the serum of patients with tuberculosis. The 65kD antigen is present in purified protein derivatives (PPD's) of M. tuberculosis, M. bovis, and other mycobacteria. Thole, J.E.R. et al., Infection Immunity, 50:800-806 (1985). Finally, helper T cell clones reactive with recombinant 65kD antigen have been isolated from patients with tuberculosis, indicating that this antigen is involved in the cell-mediated as well as the humoral immune response to tuberculosis. Among the major antigens of the leprosy bacillus, the 65kD antigen appears to elicit antibody and T cell responses similar to those observed for the M. tuberculosis antigen. Both serum antibodies and T cells directed against the 65kD M. leprae antigen have been observed in patients with leprosy. Britton, W.J. et al., Journal of Immunology, 135:4171-4177 (1985); Mustafa, A.S. et al., Nature, 319:63-66 (1986). In addition, T cell clones from leprosy patients have been found to respond to recombinant 65kD protein of M. bovis, as well as to PPD's from both M. bovis BCG and M. leprae. Emmrich, F. et al., Journal of Experimental Medicine, 163:1024-1029 (1986); Shankar, P. et al., Journal of Immunology, 136:4255-4263 (1986). It is interesting to note that in vaccine trials in Asia and Africa, BCG provided significant protection against leprosy, ranging from 20% to 80%. Fine, P., Tubercle, 65:137-153 (1984). An intriguing possibility is that the M. bovis BCG 65kD antigen is involved in engendering the immune protection provided by this vaccine against M. leprae, as well as against M. tuberculosis.

In addition to the 65kD antigen, there is evidence that the 19kD and 71kD antigens of M. tuberculosis may be particularly important in the immune response to this bacillus. Helper T cell clones from tuberculosis patients have been isolated which respond to the recombinant 19kD protein. The 71kD antigen is recognized by the humoral immune system of both mice and rabbits, and antibody to this antigen has been shown to be a prominent component of hyperimmune anti-M. tuberculosis rabbit sera.

VIII. Isolation of DNA Clones for Genes Encoding Proteins of Additional Mycobacteria

Using the procedures described above for isolation of genes encoding M. tuberculosis proteins, genes encoding proteins of additional mycobacteria were isolated. DNA clones containing genes encoding the following proteins were isolated: Mycobacterium Protein Clone M. bovis BCG 71kD PL1-101

65kD PL1-105

19kD PL1-501

14kD PL1-502

M. smegmatis 65kD PL1-206

M. avium 65kD PL1-401

M. africanum 65kD PL1-301 For purposes of comparison, genes encoding the following proteins were isolated for M. tuberculosis and M. leprae:

Mycobacterium Protein Clone

M. tuberculosis 71kD Y3272

65kD Y3150

19kD Y3147

14kD Y3248

M. leprae 65kD

The following strains were used for this purpose:

Species Isolate

M. leprae Armadillo isolate (WHO)

M. tuberculosis Erdmann strain

M. africanum African clinical isolate

M. bovis BCG Danish vaccine strain

M. smegmatis MC²-6

M. avium AIDS patient isolate

DNA from recombinant lambda gtll clones was isolated, as described above, and mapped with restriction endonucleases, using standard techniques. Davis, R.W. et al., Advanced Bacterial Genetics: A Manual for Genetic Engineering, Cold Spring Harbor (1980). Figure 4 presents a comparison of the restriction maps for four genes of M. tuberculosis with the restriction maps for four genes of M. bovis BCG which encode proteins of the same molecular weight. As is evident from the figure, in each case, the restriction sites on the two genes (e.g., those on the M. tuberculosis gene and those on the M. bovis gene which encodes a protein of the same molecular weight) are essentially identical. This indicates that the sequence of the genes of the two mycobacteria (at least those encoding these four proteins) are very similar and, therefore, the proteins they encode are also very similar.

Figure 5 presents a comparison of the restriction map for the gene encoding the 65kD protein for the six mycobacteria. As is evident, the restriction maps for the genes encoding the 65kD protein of M. tuberculosis, M. africanum, M. bovis BCG, M. smegmatis and M. avium are essentially identical. The fact that there is no detectable difference among these mycobacteria at the level of the restriction map is an indication that, at least at this level, the encoded proteins are the same.

As is also evident, the map of the M. leprae 65kD gene has several identical restriction sties in common with those of the other mycobacteria; it also has two sites not found in the other genes and lacks three sites present in the others. This indicates that, at the level of the restriction map, there are similarities in the DNA (and the encoded protein).

In addition, however, there are differences apparent at this level.

IX. Diagnostic, Therapeutic and Preventive

Applications The isolation of genes encoding major protein antigens of M. tuberculosis makes it possible to address problems which presently exist in diagnosing treating and preventing tuberculosis. Isolation of genes encoding proteins of other mycobacteria, such as M. bovis BCG, M. africanum, M. smegmatis and M. avium makes it possible to address similar problems in diseases which they cause.

The nucleotide sequence of three of the five genes has been determined. The sequence of the remaining genes can be determined using well-known methods, such as that of Sanger et al. Sanger, F. et. al., Proceedings of the National Academy of Sciences, USA, 74:5463-5467 (1977). The amino acid sequence of each of the immunodominant proteins has been deduced from the nucleotide sequence of the three genes and can be done for the others.

Identification and characterization of the genes for major tuberculosis protein antigens and of the proteins themselves make it possible to develop improved reagents for diagnosis and immunoprophylaxis of tuberculosis. Proteins antigens encoded by an entire gene, or amino acid sequences (e.g., peptides, protein fragments) which make up the antigenic determinant of a M. tuberculosis antigen (i.e., M. tuberculosis-specific antigenic determinants) may be used in serodiagnostic tests and skin tests. Such antigens would be highly specific to the tuberculosis bacillus and the tests in which they are used would also be highly specific. Highly specific serological tests would be of great value in screening populations for individuals producing antibodies to M. tuberculosis-specific antigenic determinants; in monitoring the development of active disease in individuals and in assessing the efficacy of treatment. As a result, early diagnosis of tuberculosis will be feasible, thus making it possible to institute treatment at an early stage of the disease and, in turn, to reduce the likelihood it will be transmitted.

As a result of the work described, it is also possible to determine which segment(s) of the M. tuberculosis antigen is recognized by M. tuberculosis-specific T cells. A mixture of peptides recognized by helper T cells can serve as a specific skin test antigen useful in assessing immunological status (delayed hypersensitivity) of infected individuals and those with whom they come in contact. This specific skin test antigen would be useful in evaluating rapidly the immunological efficacy of anti-tuberculosis vaccines. It is reasonable to expect that the products encoded by M. tuberculosis genes, particularly those shown to be recognized by helper T cells, are themselves immunogenic and thus useful components of vaccines against tuberculosis. These products include proteins and portions of such proteins

(e.g., polypeptides and peptides). For example, one approach to vaccine development is the introduction of genes encoding products (e.g., polypeptides) which provide immunological protection into viruses such as vaccinia virus, or bacteria, such as cultivatable mycobacteria, thus producing a vaccine capable of engendering long-lasting and very specific immunity. The genes encoding five immunodominant protein antigens of the tuberculosis bacillus, described herein, are useful for that purpose; genes encoding the 65kD, 19kD and 71kD antigens, or a portion thereof, are particularly valuable in vaccine construction.

Because of the similarities in the DNA encoding similarly-sized proteins and, thus, of the encodied proteins themselves, it is possible that, for example, a vaccine effective against two or more of the mycobacteria can be produced.

EXEMPLIFICATION

Isolation and Analysis of Recombinants Expressing the 65kD M. tuberculosis Antigen

The recombinant DNA library of M. tuberculosis genomic DNA fragments in the lambda gtll vector was constructed as described above. Recombinant phage lambda RY3143 and lambda RY3146 were used. Young, R.A. et al., Proceedings of the National Academy of Sciences, USA, 82:2583-2587 (1985). Subclones of the mycobacterial DNA inserts in these recombinant phage were constructed in pUC19 or M13mp9 vectors using standard recombinant DNA techniques. Messing, J. and J. Viera, Gene, 19:269-276 (1982). Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Monoclonal antibodies specific for the 65kD antigen were obtained from the Immunology of Tuberculosis Scientific Working Group under a grant from the WHO/World Bank/UNDP Special Program for Vaccine Development. These antibodies included

IT-13 (WTB-78), IT-31 (SA2D5H4), and IT-33 (MLIIH9). Coates, A.R.M.. et al., Lancet, 2:167-169 (1981). Gillis, T.P. and T.M. Buchanon, Immunology, 37:172-178 (1982). Anti-B-galactosidase antibodies were purchased from CooperBiomedical. Polyclonal rabbit antisera directed against a sonicate of M. tuberculosis strain H37Rv were elicited as described by Minden and co-workers. Minden, P. et al., Infect. Immun., 46:519-525 (1984). Results are shown in Table 2.

TABLE 2: PATTERNS OF ANTIBODY REACTIVITIES^a

Number of Clones Reactivity with Antibodies

IT-13 IT-31 IT-33

27 + + +

1 + + +

2 + - +

3 - + +

1 + - -

2 - + -

2 - - +

^a: Recombinant clones expressing antigens reactive with the 65kD antigen specific monoclonal antibodies IT-13, IT-31, and IT-33 were isolated as described above. For the initial screen, a pool of the three antibodies was used; it contained a 1:1000 dilution of each antibody to screen a total of about 8 × 10 5 recombinant phage from the lambda gtll-M. tuberculosis library. To determine which monoclonal antibody reacted with which of the 38 plaque-purified recombinants, about 100 pfu of each recombinant phage were inoculated in small spots on a lawn of Y1090. The phage were allowed to grow and induced to synthesize the foreign proteins as described previously. The filters were then reacted with a 1:1000 dilution of one of the monoclonal hybridoma antibodies as described above. The lambda gtll-M. tuberculosis library was screened with the monoclonal antibodies specific for the 65kD antigen and clones reactive with them were isolated essentially as described by Young et al. Young, R.A. et al., Proceedings of the National Academy of Sciences, USA, 82:2583-2587 (1985). Briefly, for each 150mm LB plate, 0.6ml of a fresh overnight culture of Y1090 was infected with 1-2 ×10⁵ plaque forming units of the library. After 3.5-4 hours of growth at 42^ºC, the plaques were overlaid with a dry nitrocellulose filter which had been saturated with 10mM isopropyl-B-D-thiogalactopyranoside (IPTG). The plates were incubated an additional 3.5-4 hours at 37^ºC and then removed to room temperature and the position of the filters marked. The filters were washed briefly in TBST (50 mM Tris-HCl, pH 8, 150mM NaCl, 0.05% Tween 20) and then incubated in TBST + 20% fetal calf serum. After 30 minutes at room temperature, the filters were transferred to TBST plus antibody. For the initial screen, the antibody mix contained a 1:1000 dilution of IT-13, IT-31, and IT-33. The filters were incubated with the antibody solution overnight at 4^ºC with gentle agitation, washed in TBST and reacted with biotinylated goat anti-mouse immunoglobulin, the Vectastain ABC reagent, and developer as described by the manufacturer (Vector Laboratories). After the color had developed the filters were washed with several changes of water and air dried. Phage corresponding to positive signals were twice plaque purified. To determine which monoclonal antibodies reacted with which of the recombinant phage, about 100 pfu of a purified phage stock were inoculated in a small spot on a lawn of Y1090 bacteria on an LB plate. The phage were allowed to grow and induced to synthesize the foreign proteins as described above. The filters were then reacted with a 1:1000 dilution of one of the monoclonal antibodies. The subsequent steps were the same as for the initial screen. Western blot assays were carried out as follows: Cells containing phage or plasmids in which the expression of the foreign sequences was under the control of the E. coli lac gene regulatory sequences were induced to synthesize the foreign proteins by incubating the cells in the presence of 2.5mM IPTG for 2 hours. Crude lysates of cells expressing lambda gtll recombinants were made as described in Huynh et al. Huynh, T.V. et al., In: DNA Cloning Techniques: A Practical Approach, (D. Glover, ed.) IRL Press, Oxford, Vol. 1, pp. 49-78 (1985). Crude lysates of cells expressing plasmid encoded proteins were made by harvesting cells from overnight cultures and resuspending the cells in 10 mM Tris pH7.5/10 mM EDTA containing 100 ug lysozyme/ml. After 10 minutes at room temperature, SDS was added to a final concentration of 0.5%. A protease inhibitor (Trasylol, Boehringer Mannheim) was added to all crude lysates at a final concentration of 0.3%. The crude protein preparations were electrophoresed on 10% polyacrylamide-SDS

Laemmli gels and the separate proteins electrophor etically transferred to nitrocellulose. Laemmli, U.K., Nature, 227:680-685 (1970). Towbin, H. et al., Proceedings of the National Academy of Sciences, USA, 76:4350-4354 (1979). The immobilized proteins were reacted with a 1:1000 dilution of monoclonal antibody IT-13 in TBST overnight at 4°C. The nitrocellulose filters were then washed, reacted with peroxidase-conjugated goat anti-mouse immuno-globulin, and developed as described by Niman and co-workers. Niman, H.L. et al., Proceedings of the National Academy of Sciences, USA, 80:4949-4953 (1983).

The sequences of 5'-end-labeled restriction fragments of the mycobacterial DNA were determined by a modification of the partial chemical degradation technique of Maxam and Gilbert. Brow, M.A.D. et al., Mol. Biol. Evol., 2:1-12 (1985). Maxam, A.M. and W. Gilbert, Proceedings of the National Academy of Sciences, USA, 74:560-564 (1976). For the M13/dideoxy sequencing studies,

Sau3AI fragments from the mycobacterial DNA inserts were subcloned into the BamHI site of M13mp9. Phage DNA was isolated from the M13 recombinants and subjected to the dideoxy chain termination sequencing reactions. Biggin, M.D. et al.,

Proceedings of the National Academy of Sciences, USA, 80:3963-3965 (1983). Sanger, F. et al., Journal of Molecular Biology, 143:161-178 (1980). The products of the sequencing reactions were electrophoresed on 6% acrylamide/7M urea/0.5-2.5 × TBE gradient sequencing gels. The gels were dried under vacuum and exposed to Kodak XRP-1 film. The nucleotide sequences were determined independently for both strands of the mycobacterial DNA.

Computer-aided analyses of the nucleic acid sequences and deduced protein sequences were performed using the Databases and programs provided by the Nucleic Acid and Protein Identification Resources of the National Institutes of Health as well as the programs of Chou and Fasman and Hopp and Woods. Chou, P.Y. and G.D. Fasman, Adv. Enzym., 47:45-148 (1978). Hopp, T.P. and K.P. Woods, Proceedings of the National Academy of Sciences, USA, 78: 3824-3828 (1981). The nucleotide sequence of the region containing the M. tuberculosis 65kD gene and the deduced amino acid sequence of the two long open reading frames are represented in Figure 8.

B-galactosidase assays were also carried out. Cells were grown in LB broth or LB broth plus 2.5mM IPTG to an OD₆₀₀ of about 0.3. Crude lysates were made and b-galactosidase activity assayed as described by Miller. Miller, J.H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972).

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 herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

1. Isolated DNA encoding an immunogenic protein antigen of Mycobacterium tuberculosis.

2. DNA of Claim 1 selected from the group consist- ing of DNA encoding Mycobacterium tuberculosis protein antigens of molecular weight 71kD, 65kD, 19kD, 14kD and 12kD.

3. Isolated DNA encoding an immunodominant protein antigen of Mycobacterium tuberculosis, the protein antigen having a molecular weight of approximately 65kD and recognized by a monoclonal antibody selected from the group consisting of: IT-31; C1.1; IIH9; IIC8; T2.3; Y1-2; SA2.D7C and IT-13.

4. Isolated DNA encoding an immunodominant protein antigen of Mycobacterium tuberculosis, the protein antigen having a molecular weight of approximately 19kD and recognized by a monoclonal antibody selected from the group consisting of: IT-10; IT-12; IT-16; and IT-19.

5. Isolated DNA encoding an immunodominant protein antigen of Mycobacterium tuberculosis, the protein antigen having a molecular weight of approximately 71kD and recognized by the monoclonal antibody IT-11.

6. Isolated DNA encoding an antigenic determinant of Mycobacterium tuberculosis protein.

7. DNA of Claim 6 which encodes an antigenic determinant selected from the group consisting of antigenic determinants of Mycobacterium tuberculosis proteins of molecular weight 71kD, 65kD, 19kD, 14kD and 12kD.

8. Isolated DNA encoding an amino acid sequence of an antigenic determinant of Mycobacterium tuberculosis protein, said protein having a molecular weight of approximately 65kD.

9. Isolated Mycobacterium tuberculosis DNA encoding an immunodominant protein antigen having a molecular weight of approximately 65kD, said DNA selected from the group consisting of: a. the DNA insert of clone Y3141; b. the DNA insert of clone Y3143; c. the DNA insert of clone Y3150; d. the DNA insert of clone Y3253; and e. the DNA insert of clone Y3262.

10. A protein antigen encoded by DNA of Claim 9.

11. A protein antigen of Claim 10, wherein the protein antigen is recognized by a monoclonal antibody selected from the group consisting of IT-31; C1.1; IIH9; IIC8; T2.3; Y1-2; SA2.D7C and IT-13.

12. Isolated DNA having a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence represented in Figure 6, or a portion thereof; b) the nucleotide sequence represented in Figure 7, or a portion thereof; and c) the nucleotide sequence represented in Figure 8, or a portion thereof.

13. A protein or a peptide selected from the group consisting of: a) proteins or peptides encoded by the nucleotide sequence represented in

Figure 6, or a portion thereof; b) proteins or peptides encoded by the nucleotide sequence represented in Figure 7, or a portion thereof; and c) proteins or peptides encoded by the nucleotide sequence represented in Figure 8, or a portion thereof.

14. A peptide having the amino acid sequence of an antigenic determinant of Mycobacterium tuberculosis protein, said antigenic determinant being unique to Mycobacterium tuberculosis protein.

15. A peptide encoded by isolated Mycobacterium tuberculosis DNA, said peptide recognized by helper T cells.

16. A peptide encoded by the Mycobacterium tuberculosis DNA insert of clone Y3150 or a portion of said DNA insert.

17. Isolated DNA encoding a protein of Mycobacterium africanum the protein having a molecular weight of 65kD.

18. Isolated DNA encoding a protein of Mycobacterium avium, the protein having a molecular weight of 65kD.

19. A vaccine comprising DNA encoding Mycobacterium tuberculosis protein in a recombinant vaccine vector, capable of expressing said DNA.

20. A vaccine of Claim 19 in which the recombinant vaccine vector is vaccinia virus or cultivat- able mycobacteria.

21. A vaccine of Claim 20 in which the DNA encodes the 65kD Mycobacterium tuberculosis protein recognized by the monoclonal antibody IT-13, or a portion of said protein.

22. A vaccine comprising DNA encoding an antigenic determinant unique to Mycobacterium tuberculosis cultivatable mycobacteria capable of expressing said DNA.

23. A method of detecting antibody against Mycobacterium tuberculosis in a biological fluid, comprising the steps of: a) incubating an immunoadsorbent comprising a solid phase to which is attached immunodeterminant Mycobacterium tuberculosis protein with a sample of the biological fluid to be tested, under conditions which allow the anti-Mycobacterium tuberculosis antibody in the sample to bind to the immunoadsorbent; b) separating the immunoadsorbent from the sample; and c) determining if antibody is bound to the immunoadsorbent, as an indication of anti-Mycobacterium tuberculosis in the sample.

24. A method of Claim 23 in which the Mycobacterium tuberculosis protein attached to the solid phase has a molecular weight of approximately 65kD.

25. A method of detecting antibody against Mycobacterium tuberculosis in a biological fluid, comprising the steps of: a) incubating an immunoadsorbent comprising a solid phase to which is attached a peptide having the amino acid sequence of an antigenic determinant of Mycobacterium tuberculosis protein with a sample of the biological fluid to be tested, under conditions which allow antibody against Mycobacterium tuberculosis to bind to the immunoadsorbent; b) separating the immunoadsorbent; and c) determining if antibody is bound to the immunoadsorbent, as an indication of the presence of the antibody against Mycobacterium tuberculosis in the sample.

26. A method of Claim 25 in which the peptide has the amino acid sequence of an antigenic determinant which is unique to Mycobacterium tuberculosis protein.

27. A kit useful in detecting antibody against Mycobacterium tuberculosis in a biological fluid, comprising a collection of reagents for immunoassay of said antibody, said collection of reagents a solid phase to which is attached immunodeterminant Mycobacterium tuberculosis protein or a peptide having the amino acid sequence of an antigenic determinant of Mycobacterium tuberculosis.