Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1999-04-07
2003-09-30
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S009200, C424S130100, C424S164100, C424S168100, C424S248100, C435S069100, C435S069300, C514S04400A, C530S350000, C536S023100, C536S023700
Reexamination Certificate
active
06627198
ABSTRACT:
1. INTRODUCTION
The present invention relates to fusion proteins containing at least two
Mycobacterium tuberculosis
antigens. In particular, it relates to bi-fusion proteins which contain two individual
M. tuberculosis
antigens, tri-fusion proteins which contain three
M. tuberculosis
antigens, tetra-fusion proteins which contain four
M. tuberculosis
antigens, and penta-fusion proteins which contain five
M. tuberculosis
antigens, and methods for their use in the diagnosis, treatment and prevention of tuberculosis infection.
2. BACKGROUND OF THE INVENTION
Tuberculosis is a chronic infectious disease caused by infection with
M. 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 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.
In order to control the spread of tuberculosis, effective vaccination and accurate early diagnosis of the disease are of utmost importance. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common Mycobacterium employed for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of
M. 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 with this agent.
Diagnosis of tuberculosis 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 induration 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 Acquired Immunodeficiency Syndrome 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, interleukin-12 (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, 1994, Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, D.C.
Accordingly, there is a need for improved vaccines, and improved methods for diagnosis, preventing and treating tuberculosis.
3. SUMMARY OF THE INVENTION
The present invention relates to fusion proteins of
M. tuberculosis
antigens. In particular, it relates to fusion polypeptides that contain two or more
M. tuberculosis
antigens, polynucleotides encoding such polypeptides, methods of using the polypeptides and polynucleotides in the diagnosis, treatment and prevention of
M. tuberculosis
infection.
The present invention is based, in part, on the inventors' discovery that polynucleotides which contain two to five
M. tuberculosis
coding sequences produce recombinant fusion proteins that retain the immunogenicity and antigenicity of their individual components. The fusion proteins described herein induced both T cell and B cell responses, as measured by T cell proliferation, cytokine production, and antibody production. Furthermore, a fusion protein was used as an immunogen with adjuvants in vivo to elicit both cell-mediated and humoral immunity to
M. tuberculosis
. Additionally, a fusion protein was made by a fusion construct and used in a vaccine formulation with an adjuvant to afford long-term protection in animals against the development of tuberculosis. The fusion protein was a more effective immunogen than a mixture of its individual protein components.
In a specific embodiment of the invention, the isolated or purified
M. tuberculosis
polypeptides of the invention may be formulated as pharmaceutical compositions for administration into a subject in the prevention and/or treatment of
M. tuberculosis
infection. The immunogenicity of the fusion protein may be enhanced by the inclusion of an adjuvant.
In another aspect of the invention, the isolated or purified polynucleotides are used to produce recombinant fusion polypeptide antigens in vitro. Alternatively, the polynucleotides may be administered directly into a subject as DNA vaccines to cause antigen expression in the subject, and the subsequent induction of an anti-
M. tuberculosis
immune response.
It is also an object of the invention that the polypeptides be used in in vitro assays for detecting humoral antibodies or cell-mediated immunity against
M. tuberculosis
for diagnosis of infection or monitor of disease progression. Additionally, the polypeptides may be used as an in vivo diagnostic agent in the form of an intradermal skin test. Alternatively, the polypeptides may be used as immunogens to generate anti-
M tuberculosis
antibodies in a non-human animal. The antibodies can be used to detect the target antigens in vivo and in vitro.
REFERENCES:
patent: 5330754 (1994-07-01), Kapoor et al.
patent: WO 95/01440 (1995-01-01), None
patent: WO 95/14713 (1995-06-01), None
patent: WO 97/09428 (1997-03-01), None
patent: WO 97/09429 (1997-03-01), None
Pal et al., “Immunization with Extracellular Proteins ofMycobacterium tuberculosisInduces Cell-Mediated Immune Responses and Substantial Protective Immunity in a Guinea Pig Model of Pulmonary Tuberculosis”;Infection and Immunityvol. 60, No. 11, pp. 4781-4792 (Nov. 1992).
Philipp et al., An integrated map of the genome of the tubercle bacillusMycobacterium tuberculosisH37Rv, and comparison withMycobacterium leprae, Proc. Natl. Acad. Sci, 93:3132-3137 (1996).
Lee et al., Characterization of the Major Membrane Protein of VirulentMycobacterim tuberculosis, Infection and Immunity, p. 2066-2074 (5/92).
Alderson Mark
Campos-Neto Antonio
Dillon Davin C.
Reed Steven G.
Skeiky Yasir A.
Corixa Corporation
Swartz Rodney P
Townsend and Townsend and Crew
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