Methods for detecting Mycobacterium tuberculosis disease
Inventors
Lewinsohn, Deborah A. • Lewinsohn, David M.
Assignees
Oregon Health and Science University • US Department of Veterans Affairs
Publication Number
US-8658350-B2
Publication Date
2014-02-25
Expiration Date
2029-09-22
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Abstract
Methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in a subject are disclosed, wherein the subject is a child, a subject with a latent Mycobacterium tuberculosis infection. Method are also disclose for detecting an extra-pulmonary Mycobacterium tuberculosis infection in a subject. The methods include detecting the presence of CD8+ T cells that specifically recognize an Mtb polypeptide. The methods include in vitro assays for detecting the presence of CD8+ T cells in a biological sample.
Core Innovation
Methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in subjects, particularly children and individuals with latent tuberculosis infection (LTBI), are disclosed. The methods focus on detecting the presence of CD8+ T cells that specifically recognize Mtb polypeptides, through in vitro assays involving biological samples and optionally in vivo assays detecting delayed type hypersensitivity reactions in the subject.
The problem addressed arises from the difficulty in diagnosing tuberculosis in children and detecting latent Mtb infections. Children are disproportionately susceptible to TB, often developing severe forms, yet commonly present with negative traditional diagnostic markers such as sputum acid-fast bacillus smears. Current diagnostic methods, like the tuberculin skin test (TST), lack the sensitivity and specificity needed to distinguish infected from vaccinated individuals and perform poorly in children and latent infections. There is thus a need for improved diagnostic methods that can accurately detect active and latent tuberculosis, including extra-pulmonary infections, especially in vulnerable populations such as children and immunocompromised individuals.
Claims Coverage
The patent contains 26 claims focused on methods for diagnosing tuberculosis in children by detecting CD8+ T cell responses to specific Mycobacterium polypeptides, with inventive features centered on T cell isolation, detection, and specific polypeptides.
Detection of tuberculosis in children by identifying CD8+ T cells recognizing Mtb polypeptides
The method involves isolating CD8+ T cells from a biological sample of a human child suspected of tuberculosis, contacting the cells with one or more Mycobacterium polypeptides, and determining if the CD8+ T cells specifically recognize these polypeptides, thereby indicating tuberculosis disease in the child.
Use in young and infant children suspected of pulmonary or extra-pulmonary tuberculosis disease
The methods are applicable to children less than five years old, infants, prepubescent children, and include detection of both pulmonary and various extra-pulmonary tuberculosis diseases such as lymphadenitis, pleural tuberculosis, bone and joint tuberculosis, central nervous system tuberculosis, abdominal tuberculosis, miliary tuberculosis, and tuberculous pericarditis.
Detection of CD8+ T cell activation via cytokine expression
Determining specific recognition of the Mtb polypeptides by CD8+ T cells includes measuring cytokine expression, particularly interferon-γ (IFN-γ), using antibodies that specifically bind IFN-γ.
Use of specific Mtb polypeptides and peptides for diagnosis
The Mycobacterium polypeptides used comprise the amino acid sequence of SEQ ID NO: 61 or consecutive 9 to 20 amino acids thereof that specifically bind major histocompatibility complex (MHC) class I, including sequences set forth as SEQ ID NOs: 62-83.
Use of diverse biological samples and in vitro culture for assay
The biological samples include blood, isolated peripheral blood mononuclear cells, sputum, lung biopsy, lymph node biopsy, saliva, cerebrospinal fluid, or isolated CD3+ T cells. The CD8+ T cells can be cultured in vitro with the Mycobacterium polypeptides to enhance detection.
Additional detection methods including delayed type hypersensitivity and antigen presence
The methods further encompass detecting a delayed type hypersensitivity reaction to Mtb and detecting the presence of Mycobacterium polypeptides or encoding polynucleotides in the subject's sample, the latter via antibodies or polymerase chain reaction assays.
Detection of CD8+ T cells using labeled MHC class I tetramer complexes
A method is disclosed for detecting CD8+ T cells that bind a Mycobacterium polypeptide, by contacting peripheral blood mononuclear cells with a reagent comprising a Mycobacterium polypeptide (specifically 9-20 amino acids of SEQ ID NO: 61 that bind MHC class I), HLA heavy chain polypeptide, β2-microglobulin, and streptavidin, with detection of reagent binding indicating specific T cells.
The claims cover diagnostic methods for tuberculosis in children employing specific detection of CD8+ T cell responses to defined Mycobacterium tuberculosis polypeptides, particularly CFP-10 (SEQ ID NO: 61) and its epitopes, using cytokine assays, tetramer staining, and molecular detection to achieve specific and sensitive identification of tuberculosis disease including pulmonary and extra-pulmonary forms.
Stated Advantages
Improved sensitivity and specificity for detecting tuberculosis disease in children, including difficult cases such as extra-pulmonary tuberculosis.
Ability to distinguish latent tuberculosis infection and active tuberculosis disease.
Methods that overcome limitations of traditional skin tests, such as inability to differentiate between BCG vaccination and active infection, and poor performance in children.
Diagnostic methods that enable early detection essential for prompt intervention in children who are at higher risk of severe forms of tuberculosis.
Applicability to immunocompromised subjects and various forms of tuberculosis disease, including pulmonary and extra-pulmonary manifestations.
Documented Applications
Diagnosing tuberculosis disease in children, including those less than five years old and infants.
Detecting latent Mycobacterium tuberculosis infection in subjects.
Diagnosing extra-pulmonary tuberculosis disease in children and adults.
Monitoring progression of tuberculosis infection and assessing effectiveness of therapeutic regimens by measuring CD8+ T cell responses.
Confirmatory testing of tuberculosis infection using in vivo delayed type hypersensitivity skin tests based on Mtb polypeptides.
Detecting antibodies against Mycobacterium tuberculosis polypeptides in biological samples as a confirmatory diagnostic assay.
Detecting Mycobacterium tuberculosis nucleic acids in biological samples as part of a confirmatory diagnosis.
Use of tetrameric MHC class I-peptide complexes to identify antigen-specific CD8+ T cells in blood samples.
Evaluating vaccine efficacy and immune response in animal models by measuring CD8+ T cell responses to specific Mtb polypeptides.
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