Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Parasitic organism or component thereof or substance...
Reexamination Certificate
1997-12-18
2001-05-08
Graser, Jennifer (Department: 1641)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Parasitic organism or component thereof or substance...
C424S185100, C424S191100, C424S192100, C424S193100, C424S200100, C435S007100, C435S007220, C435S004000, C435S007400, C435S007920, C435S069100, C435S069300, C435S069700, C435S071100, C530S300000, C530S324000, C530S333000, C530S334000, C530S344000, C530S403000, C530S412000, C530S350000
Reexamination Certificate
active
06228372
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the diagnosis of
T. cruzi
infection. The invention is more particularly related to the use of one or more
T. cruzi
antigenic peptides, or antibodies thereto, in methods and diagnostic kits to screen individuals and blood supplies for
T. cruzi
infection. The invention is also directed to vaccine compositions for immunizing an individual to prevent Chagas' disease.
BACKGROUND OF THE INVENTION
Protozoan parasites are a serious health threat in many areas of the world.
Trypanosoma cruzi
(
T. cruzi
) is one such parasite that infects millions of individuals, primarily in Central and South America. Infections with this parasite can cause Chagas' disease, which may result in chronic heart disease and a variety of immune system disorders. It is estimated that 18 million people in Latin America are infected with
T. cruzi,
but there is no reliable treatment for the clinical manifestations of infection. No vaccine for the prevention of Chagas' disease is currently available.
The most significant route of transmission in areas where the disease is endemic is through contact with an infected triatomid bug. In other areas, however, blood transfusions are the dominant means of transmission. To inhibit the transmission of
T. cruzi
in such regions, it is necessary to develop accurate methods for diagnosing
T. cruzi
infection in individuals and for screening blood supplies. Blood bank screening is particularly important in South America, where 0.1%-62% of samples may be infected and where the parasite is frequently transmitted by blood transfusion. There is also increasing concern that the blood supply in certain U.S. cities may be contaminated with
T. cruzi
parasites.
The diagnosis of
T. cruzi
infection has been problematic, since accurate methods for detecting the parasite that are suitable for routine use have been unavailable. During the acute phase of infection, which may last for decades, the infection may remain quiescent and the host may be asymptomatic. As a result, serological tests for
T. cruzi
infection are the most reliable and the most commonly used.
Such diagnoses are complicated, however, by the complex life cycle of the parasite and the diverse immune responses of the host. The parasite passes through an epimastigote stage in the insect vector and two main stages in the mammalian host. One host stage is present in blood (the trypomastigote stage) and a second stage is intracellular (the amastigote stage). The multiple stages result in a diversity of antigens presented by the parasite during infection. In addition, immune responses to protozoan infection are complex, involving both humoral and cell-mediated responses to the array of parasite antigens.
While detecting antibodies against parasite antigens is the most common and reliable method of diagnosing clinical and subclinical infections, current tests are expensive and difficult. Most serological tests use whole or lysed
T. cruzi
and require positive results on two of three tests, including complement fixation, indirect immunofluorescence, passive agglutination or ELISA, to accurately detect
T. cruzi
infection. The cost and difficulty of such tests has prevented the screening of blood or sera in many endemic areas.
Accordingly, there is a need in the art for more specific and sensitive methods of detecting
T. cruzi
infections in blood supplies and individuals. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, this invention provides compounds and methods for detecting and protecting against
T. cruzi
infection in individuals and in blood supplies, and for screening for
T. cruzi
infection in biological samples. In one aspect, the present invention provides methods for detecting
T. cruzi
infection in a biological sample, comprising (a) contacting the biological sample with a polypeptide comprising an epitope of a
T. cruzi
antigen having an amino acid sequence encoded by a nucleotide sequence recited in SEQ ID NO: 1-SEQ ID NO:22, or a variant of such an antigen that differs only in conservative substitutions and/or modifications; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide, therefrom detecting
T. cruzi
infection in the biological sample.
In another aspect of this invention, polypeptides are provided comprising an epitope of a
T. cruzi
antigen having an amino acid sequence encoded by a nucleotide sequence recited in SEQ ID NO: 1-SEQ ID NO:21, or a variant of such an antigen that differs only in conservative substitutions and/or modifications.
Within related aspects, DNA sequences encoding the above polypeptides, 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 diagnostic kits for detecting
T. cruzi
infection in a biological sample, comprising (a) a polypeptide comprising an epitope of a
T. cruzi
antigen having an amino acid sequence encoded by a nucleotide sequence recited in SEQ ID NO: 1-SEQ ID NO:22, or a variant of such an antigen that differs only in conservative substitutions and/or modifications; and (b) a detection reagent.
In yet another aspect of the invention, methods for detecting the presence of
T. cruzi
infection in a biological sample are provided, comprising (a) contacting a biological sample with a monoclonal antibody that binds to an epitope of a
T. cruzi
antigen having an amino acid sequence encoded by a nucleotide sequence recited in SEQ ID NO :1-SEQ ID NO:22, or a variant of such an antigen that differs only in conservative substitutions and/or modifications; and (b) detecting in the biological sample the presence of
T. cruzi
parasites that bind to the monoclonal antibody.
Within related aspects, pharmaceutical compositions comprising the above polypeptides and a physiologically acceptable carrier, and vaccines comprising the above polypeptides in combination with an adjuvant, are also provided.
The present invention also provides, within other aspects, methods for inducing protective immunity against Chagas' disease in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above.
Within other aspects, the present invention provides methods for detecting
T. cruzi
infection in a biological sample, comprising (a) contacting the biological sample with a first polypeptide comprising an epitope of a
T. cruzi
antigen having an amino acid sequence encoded by a nucleotide sequence recited in SEQ ID NO:1-SEQ ID NO:22, or a variant of said antigen that differs only in conservative substitutions and/or modifications; (b) contacting the biological sample with one or more additional polypeptides comprising one or more epitopes of other
T. cruzi
antigens, or a variant thereof that differs only in conservative substitutions and/or modifications; and (c) detecting in the biological sample the presence of antibodies that bind to one or more of said polypeptides, therefrom detecting
T. cruzi
infection in the biological sample. In one embodiment, the additional polypeptide comprises an epitope of TcD, or a variant thereof that differs only in conservative substitutions and/or modifications. In another embodiment, the additional polypeptides comprise an epitope of TcD (or a variant thereof that differs only in conservative substitutions and/or modifications) and an epitope of TcE (or a variant thereof that differs only in conservative substitutions and/or modifications). In yet another embodiment, the additional polypeptides comprise an epitope of TcD (or a variant thereof that differs only in conservative substitutions and/or modifications) and PEP-2 (or a variant thereof that differs only in conservative substitutions and/or modifications).
In yet further aspects, the present invention provides combination polypeptides comprising two or more polypeptides, each polypeptide comp
Houghton Raymond L.
Lodes Michael J.
McNeill Patricia D.
Reed Steven G.
Skeiky Yasir A. W.
Corixa Corporation
Graser Jennifer
Seed Intellectual Property Law Group
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