Compounds and methods for diagnosis of tuberculosis

Details Compounds and methods for diagnosis of tuberculosis Compounds and methods for diagnosis of tuberculosis

1998-05-05

2002-10-01

Swartz, Rodney P. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S130100, C424S139100, C424S150100, C424S184100, C424S185100, C424S234100, C435S006120, C530S300000, C530S350000

Reexamination Certificate

active

06458366

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to the detection of
Mycobacterium tuberculosis
infection. The invention is more particularly related to polypeptides comprising a
Mycobacterium tuberculosis
antigen, or a portion or other variant thereof, and the use of such polypeptides for the serodiagnosis of
Mycobacterium tuberculosis
infection.
BACKGROUND OF THE INVENTION
Tuberculosis is a chronic, infectious disease, that is generally caused by infection with
Mycobacterium tuberculosis
. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as an acute inflammation of the lungs, resulting in fever and a nonproductive cough. If left untreated, serious complications and death typically result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behavior is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistance.
Inhibiting the spread of tuberculosis will require effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common Mycobacterium for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of
Mycobacterium bovis
. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable incubation at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
While macrophages have been shown to act as the principal effectors of
M. tuberculosis
immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against
M. tuberculosis
infection is illustrated by the frequent occurrence of
M. tuberculosis
in AIDS patients, due to the depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been shown to be potent producers of gamma-interferon (IFN-&ggr;), which, in turn, has been shown to trigger the anti-mycobacterial effects of macrophages in mice. While the role of IFN-&ggr; in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-&ggr; or tumor necrosis factor-alpha, activates human macrophages to inhibit
M. tuberculosis
infection. Furthermore, it is known that IFN-&ggr; stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to
M. tuberculosis
infection. For a review of the immunology of
M. tuberculosis
infection see Chan and Kaufmann, in
Tuberculosis: Pathogenesis, Protection and Control
, Bloom (ed.), ASM Press, Washington, D.C., 1994.
Accordingly, there is a need in the art for improved diagnostic methods for detecting tuberculosis. The present invention fulfills this need and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods for diagnosing tuberculosis. In one aspect, polypeptides are provided comprising an antigenic portion of a soluble
M. tuberculosis
antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications. In one embodiment of this aspect, the soluble antigen has one of the following N-terminal sequences:
(a) Asp-Pro-Val-Asp-Ala-Val-Ile-Asn-Thr-Thr-Cys-Asn-Tyr-Gly-Gln-Val-Val-Ala-Ala-Leu (SEQ ID NO: 115);
(b) Ala-Val-Glu-Ser-Gly-Met-Leu-Ala-Leu-Gly-Thr-Pro-Ala-Pro-Ser (SEQ ID NO: 116);
(c) Ala-Ala-Met-Lys-Pro-Arg-Thr-Gly-Asp-Gly-Pro-Leu-Glu-Ala-Ala-Lys-Glu-Gly-Arg (SEQ ID NO: 117);
(d) Tyr-Tyr-Trp-Cys-Pro-Gly-Gln-Pro-Phe-Asp-Pro-Ala-Trp-Gly-Pro (SEQ ID NO: 118);
(e) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa-Ala-Val (SEQ ID NO: 119);
(f) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val-Pro (SEQ ID NO: 120);
(g) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Thr-Ala-Ala-Ser-Pro-Pro-Ser (SEQ ID NO: 121);
(h) Ala-Pro-Lys-Thr-Tyr-Xaa-Glu-Glu-Leu-Lys-Gly-Thr-Asp-Thr-Gly (SEQ ID NO: 122);
(i) Asp-Pro-Ala-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala-Gln-Leu-Thr-Ser-Leu-Leu-Asn-Ser-Leu-Ala-Asp-Pro-Asn-Val-Ser-Phe-Ala-Asn (SEQ ID NO: 123);
(j) Xaa-Asp-Ser-Glu-Lys-Ser-Ala-Thr-Ile-Lys-Val-Thr-Asp-Ala-Ser; (SEQ ID NO: 129)
(k) Ala-Gly-Asp-Thr-Xaa-Ile-Tyr-Ile-Val-Gly-Asn-Leu-Thr-Ala-Asp; (SEQ ID NO: 130) or
(l) Ala-Pro-Glu-Ser-Gly-Ala-Gly-Leu-Gly-Gly-Thr-Val-Gln-Ala-Gly; (SEQ ID NO: 131)
wherein Xaa may be any amino acid.
In a related aspect, polypeptides are provided comprising an immunogenic portion of an
M. tuberculosis
antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications, the antigen having one of the following N-terminal sequences:
(m) Xaa-Tyr-Ile-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val-Pro-Gly-Lys-Ile-Asn-Val-His-Leu-Val; (SEQ ID NO: 132) or
(n) Asp-Pro-Pro-Asp-Pro-His-Gln-Xaa-Asp-Met-Thr-Lys-Gly-Tyr-Tyr-Pro-Gly-Gly-Arg-Arg-Xaa-Phe; (SEQ ID NO: 124)
wherein Xaa may be any amino acid.
In another embodiment, the soluble
M. tuberculosis
antigen comprises an amino acid sequence encoded by a DNA sequence selected from the group consisting of the sequences recited in SEQ ID NOS: 1, 2, 4-10, 13-25, 52, 94 and 96, the complements of said sequences, and DNA sequences that hybridize to a sequence recited in SEQ ID NOS: 1, 2, 4-10, 13-25, 52, 94 and 96 or a complement thereof under moderately stringent conditions.
In a related aspect, the polypeptides comprise an antigenic portion of a
M. tuberculosis
antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications, wherein the antigen comprises an amino acid sequence encoded by a DNA sequence selected from the group consisting of the sequences recited in SEQ ID NOS: 26-51, 133, 134, 158-178, 184-188, 194-196, 198, 210-220, 232, 234, 235, 237-242, 248-251, 256-271, 287, 288, 290-293 and 298-337, the complements of said sequences, and DNA sequences that hybridize to a sequence recited in SEQ ID NOS: 26-51, 133, 134, 158-178, 184-188, 194-196, 198, 210-220, 232, 234, 235, 237-242, 248-251, 256-271, 287, 288, 290-293 and 298-337, or a complement thereof under moderately stringent conditions.
In related aspects, DNA sequences encoding the above polypeptides, recombinant expression vectors comprising these DNA sequences and host cells transformed or transfected with such expression vectors are also provided.
In another aspect, the present invention provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, an inventive polypeptide and a known
M. tuberculosis
antigen.
In further aspects of the subject invention, methods and diagnostic kits are provided for detecting tuberculosis in a patient. The methods comprise: (a) contacting a biological sample with at least one of the above polypeptides; and (b) detecting in the sample the presence of antibodies that bind to the polypeptide or polypeptides, thereby detecting
M. tuberculosis
infection in the biological sample. Suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine. The diagnostic kits comp

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