Proteins expressed by Mycobacterium tuberculosis and not by BCG and their use as diagnostic reagents and vaccines

Abstract

The present invention is directed to reagents useful for generating immune responses to Mycobacterium tuberculosis and for diagnosing infection and disease in a subject that has been exposed to M. tuberculosis.

Claims

1. An in vivo method of diagnosis that discriminates between infection by Mycobacterium tuberculosis-complex and vaccination by Bacille Calmette Guerin (BCG) strain of Mycobacterium bovis in a subject comprising: administering intradermally to the subject a diagnostic reagent, wherein the diagnostic reagent comprises at least two different antigens having Mycobacterium tuberculosis-complex specific immunogenic properties, wherein the at least two antigens are polypeptides corresponding to polypeptides or antigenic fragments of polypeptides of the Mycobacterium tuberculosis-complex that are not encoded by BCG, including at least one polypeptide selected from the group consisting of (i) a first polypeptide containing MTBN4 (SEQ ID NO: 4), (ii) a second polypeptide containing an antigenic segment of MTBN4 (SEQ ID NO: 4), (iii) a third polypeptide that is identical to said first polypeptide but wherein MTBN4 has conservative substitutions and retains Mycobacterium tuberculosis-complex specific antigenic properties; and (iv) a fourth polypeptide that is identical to said second polypeptide but wherein the antigenic segment of MTBN4 has conservative substitutions and retains Mycobacterium tuberculosis-complex specific antigenic properties; and determining whether or not there has been an immune response to said diagnostic reagent.

2. The method of claim 1, wherein the immune response is a delayed-type hypersensitivity response.

3. The method of claim 2, wherein the delayed-type hypersensitivity response is reddening or swelling of the skin.

4. The method of claim 1, wherein the corresponding polypeptides or antigenic fragments of polypeptides of the Mycobacterium tuberculosis-complex that are not encoded by BCG are encoded within the RD1, RD2 and RD3 regions.

5. The method of claim 4, wherein the immune response is a delayed-type hypersensitivity response.

6. The method of claim 5, wherein the delayed-type hypersensitivity response is reddening or swelling of the skin.

7. The method of claim 1, wherein the diagnostic reagent further comprises a pharmaceutically acceptable diluent or filler.

8. The method of claim 1, wherein each of said polypeptides is recombinantly produced or chemically synthesized.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B are a depiction of the amino acid sequences of M. tuberculosis polypeptides MTBN1-MTBN8 (SEQ ID NOS:1-8, respectively).

(2) FIGS. 2A-2E are a depiction of the nucleotide sequences of the coding regions (mtbn1-mtbn8) encoding MTBN1-MTBN8 (SEQ ID NOS:9-16, respectively).

(3) FIG. 3 is a bar graph showing the delayed-type hypersensitivity responses induced by intradermal injection of 3 different test reagents (PPD, MPT64 and MTBN4) in female guinea pigs that had been either infected with M. tuberculosis cells or sensitized with BCG or M. avium cells. Group 1 animals (M. tuberculosis-infected, solid bars) showed a significant response with all three test substances. Group 2 animals (BCG-sensitized, open bars) showed a significant response to PPD and MPT64 but not MTBN4. Group 3 animals (M. avium-sensitized, hatched bars) showed a significant response to PPD only.

DETAILED DESCRIPTION

(4) The genome of M. tuberculosis [Cole et al. (1998) Nature 393: 537-544] contains open reading frames (ORFs) that have been deleted from the avirulent BCG strain.

(5) The polypeptides encoded by these ORFs are designated herein M. tuberculosis BCG Negative polypeptides (MTBN) and the ORFs are designated mtbn. The invention is based on the discovery that a MTBN polypeptide (MTBN4) elicited a skin response in animals infected with M. tuberculosis, but not in animals sensitized to either BCG or M. avium, a non-M. tuberculosis-complex strain of mycobacteria (see Example 1 below). These findings indicate that MTBN (e.g., MTBN1-MTBN8) can be used in diagnostic tests that discriminate infection of a subject by M. tuberculosis from exposure to both mycobacteria other than the M. tuberculosis-complex and BCG. The M. tuberculosis-complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Thus they can be used to discriminate subjects exposed to M. tuberculosis, and thus potentially having or being in danger of having tuberculosis, from subjects that have been vaccinated with BCG, the most widely used tuberculosis vaccine. Diagnostic assays that are capable of such discrimination represent a major advance that will greatly reduce wasted effort and consequent costs resulting from further diagnostic tests and/or therapeutic procedures in subjects that have given positive results in less discriminatory diagnostic tests.

(6) Furthermore, the results in Example 1 show that MTBN4, as expressed by whole viable M. tuberculosis organisms, is capable of inducing a strong immune response in subjects infected with the organisms and thus has the potential to be a vaccine.

(7) The MTBN polypeptides of the invention include, for example, polypeptides encoded within the RD1, RD2, and RD3 regions of the M. tuberculosis genome [Mahairas et al. (1996) J. Bacteriol. 178: 1274-1282]. Of particular interest are polypeptides encoded by ORFs within the RD1 region of the M. tuberculosis genome. However, the invention is not restricted to the RD1, RD2, and RD3 region encoded polypeptides and includes any polypeptides encoded by ORFs contained in the genome of one or more members of the M. tuberculosis genome and not contained in the genome of BCG. The amino acid sequences of MTBN1-MTBN8 are shown in FIGS. 1A and 1B and the nucleotide sequences of mtbn1-mtbn8 are shown in FIGS. 2A-2E.

(8) The invention encompasses: (a) isolated DNA molecules containing mtbn sequences (e.g., mtbn1-mtbn8) encoding MTBN polypeptides (e.g., MTBN1-MTBN8) and isolated portions of such DNA molecules that encode polypeptide segments having antigenic and immunogenic properties (i.e., functional segments); (b) the MTBN polypeptides themselves (e.g., MTBN1-MTBN8) and functional segments of them; (c) antibodies (including antigen binding fragments, e.g., F (ab) 2, Fab, Fv, and single chain Fv fragments of such antibodies) that bind to the MTBN polypeptides (e.g., MTBN1-MTBN8) and functional segments; (d) nucleic acid molecules (e.g., vectors) containing and capable of expressing one or more of the mtbn (e.g., mtbn1-mtbn8) sequences and portions of DNA molecules; (e) cells (e.g., bacterial, yeast, insect, or mammalian cells) transformed by such vectors; (f) compositions containing vectors encoding one or more M. tuberculosis polypeptides (or functional segments) including both the MTBN (e.g., MTBN1-MTBN8) polypeptides (or functional segments thereof) and previously described M. tuberculosis polypeptides such as ESAT-6.14 kDa antigen, MPT63, 19 kDa antigen, MPT64, MPT51, MTC28, 38 kDa antigen, 45/47 kDa antigen, MPB70, Ag85 complex, MPT53, and KatG (see also U.S. Pat. No. 6,087,163); (g) compositions containing one or more M. tuberculosis polypeptides (or functional segments), including both the polypeptides of the invention and previously described M. tuberculosis polypeptides such as those described above; (h) compositions containing one or more of the antibodies described in (c); (i) methods of diagnosis involving either (1) administration (e.g., intradermal injection) of any of the above polypeptide compositions to a subject suspected of having or being susceptible to M. tuberculosis infection, (2) in vitro testing of lymphocytes (B-lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes) from such a subject for responsiveness (e.g., by measuring cell proliferation, antibody production, cytokine production, or CTL activity) to any of the above polypeptide compositions, (3) testing of a bodily fluid (e.g., blood, saliva, plasma, serum, urine, or semen or a lavage such as a bronchoalveolar lavage, a vaginal lavage, or lower gastrointestinal lavage) for antibodies to the MTBN polypeptides (e. g., MTBN1-MTBN8) or functional segments thereof, or the above-described polypeptide compositions; (4) testing of a bodily fluid (e.g., as above) for the presence of M. tuberculosis, MTBN (e.g., MTBN1-MTBN8) polypeptides or functional segments thereof, or the above-described polypeptide compositions in assays using the antibodies described in (c); and (5) testing of a tissue (e.g., lung or bronchial tissue) or a body fluid (e.g., as above) for the presence of nucleic acid molecules (e.g., DNA or RNA) encoding MTBN polypeptides (e.g., MTBN1-MTBN8) (or portions of such a nucleic acid molecules) using nucleic acid probes or primers having nucleotide sequences of the nucleic molecules, portions of the nucleic molecules, or the complements of such molecules; and (j) methods of vaccination involving administration to a subject of the compositions of either (f), (g), (h) or a combination of any two or even all 3 compositions.

(9) With respect to diagnosis, purified MTBN proteins, functional segments of such proteins, or mixtures of proteins and/or the functional fragments have the above-described advantages of discriminating infection by M. tuberculosis from either infection by other bacteria, and in particular, non-pathogenic mycobacteria, or from exposure (by, for example, vaccination) to BCG.

(10) Furthermore, compositions containing the proteins, functional segments of the proteins, or mixtures of the proteins and/or the functional segments allows for improved quality control since batch-to-batch variability is greatly reduced in comparison to complex mixtures such as purified protein derivative (PPD) of tuberculin.

(11) The use of the above-described polypeptide and nucleic acid reagents for vaccination also provides for highly specific and effective immunization. Since the virulent M. tuberculosis polypeptides encoded by genes absent from avirulent BCG are likely to be mediators of virulence, immunity directed to them can be especially potent in terms of protective capacity. Where vaccination is performed with nucleic acids both in vivo and ex vivo methods can be used. In vivo methods involve administration of the nucleic acids themselves to the subject and ex vivo methods involve obtaining cells (e.g., bone marrow cells or fibroblasts) from the subject, transducing the cells with the nucleic acids, preferably selecting or enriching for successfully transduced cells, and administering the transduced cells to the subject. Alternatively, the cells that are transduced and administered to the subject can be derived from another subject. Methods of vaccination and diagnosis are described in greater detail in U.S. Pat. No. 6,087,163, the disclosure of which is incorporated herein by reference in its entirety.

(12) The following example is meant to illustrate, not limit the invention.

Example 1. MTBN4 Elicits a Specific Skin Reaction in Guinea Pigs Infected with M. tuberculosis

(13) Four groups of outbred female guinea pigs (18 per group) were used to test the usefulness of the MTBN4 polypeptide as a M. tuberculosis-specific diagnostic reagent. The four groups were treated as follows.

(14) Group 1 animals were infected by aerosol with approximately 100 M. tuberculosis strain H37Rv cells.

(15) Group 2 animals were sensitized intradermally with 106 live M. bovis BCG Japanese cells.

(16) Group 3 animals were sensitized intradermally with 106 live M. avium cells.

(17) Group 4 animals were mock-sensitized by intradermal injection with saline.

(18) Seven weeks after infection or sensitization, the animals were injected intradermally with 1 g of PPD (6 animals from each group), 2 g of purified recombinant MPT64 (6 animals from each group), or 2 g of MTBN4 (6 animals from each group). The diameter of the resulting erythema was measured 24 hours later. Data are expressed as mean diameter of erythema (in mm) and standard deviations are indicated (FIG. 3).

(19) No erythema was detected in the group 4 animals with any test substance and thus no data are shown for this group. On the other hand, group 1 animals (solid bars) showed a significant response with all three test substances. Group 2 animals (open bars) showed a significant response to PPD and MPT64 but not MTBN4.

(20) Group 3 animals showed a significant response to PPD only (hatched bars).

(21) Thus, PPD which contains antigenic/immunogenic molecules common to the M. tuberculosis-complex as well as other mycobacterial strains, gave the least discriminatory results in that it induced responses in animals infected with or sensitized to mycobacteria of the M. tuberculosis-complex (M. tuberculosis and BCG) as well as another non-pathogenic mycobacterium (M. avium).

(22) While MPT64, which is encoded and expressed by both M. tuberculosis and BCG, did not elicit a response in animals infected with M. avium, it did elicit responses in both the M. tuberculosis infected and the BCG sensitized animals. Finally, MTBN4 elicited a response in only the M. tuberculosis infected animals. Thus, it induced the most specific response and, most importantly, allowed for discrimination between animals infected with M. tuberculosis and those sensitized with BCG.

(23) Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.