Methods for detecting a Mycobacterium tuberculosis infection
Inventors
Lewinsohn, David M. • Lewinsohn, Deborah A.
Assignees
US Department of Veterans Affairs
Publication Number
US-9040233-B2
Publication Date
2015-05-26
Expiration Date
2027-03-14
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Abstract
Methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in a subject are disclosed. 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, and in vivo assays that detect a delayed type hypersensitivity reaction. The methods can also include detecting Mtb polypeptides and polynucleotides. Reagents for the detection of an Mtb infection are also disclosed.
Core Innovation
The invention discloses methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in a subject by detecting the presence of T cells, particularly CD8+ T cells, that specifically recognize an Mtb polypeptide. These methods include in vitro assays using biological samples comprising T cells and in vivo assays detecting delayed type hypersensitivity reactions. The detection can also extend to Mtb polypeptides and polynucleotides, with reagents described for diagnosing Mtb infection.
The problem addressed by the invention arises from limitations in current tuberculosis diagnostic tests, such as the tuberculin skin test, which lacks ideal sensitivity and specificity, especially in distinguishing BCG-vaccinated individuals from those infected with Mtb. Tuberculosis remains a major global health issue affecting one third of the world's population, with concerns over increasing incidence, especially among immunocompromised patients, and the emergence of multidrug-resistant strains. Effective early diagnosis is critical for disease control, and existing vaccines like BCG have inadequate efficacy or are controversial in use, highlighting the need for improved diagnostic methods.
Claims Coverage
The patent contains several independent claims focusing on methods for detecting Mycobacterium tuberculosis infection by detecting T cell responses to specific Mtb polypeptides, particularly those comprising the amino acid sequence of SEQ ID NO: 11, and uses assays to detect interferon-γ secretion or other T cell activation markers.
Detection of Mtb infection using enzyme-linked immunospot assay targeting T cells recognizing SEQ ID NO: 11
A method for detecting Mtb in a subject that comprises contacting a biological sample containing T cells with one or more isolated Mtb polypeptides, where one polypeptide includes the amino acid sequence set forth as SEQ ID NO: 11, followed by performing an enzyme-linked immunospot assay detecting interferon-γ to determine if T cells specifically recognize the polypeptide; a positive result indicates Mtb infection.
In vitro culture and detection of T cell responses to isolated Mtb polypeptides including SEQ ID NO: 11
Methods where T cells isolated from a biological sample, including CD8+ T cells, are cultured with antigen-presenting cells presenting one or more Mtb polypeptides comprising SEQ ID NO: 11 at specified concentrations for specified incubation times, followed by assays measuring T cell recognition through proliferation, cytolytic activity, or cytokine secretion such as interferon-γ, interleukin-2 or tumor necrosis factor alpha.
Detection of Mtb infection via ex vivo incubation with antigen-presenting cells and interferon-γ capture and detection
A method whereby a biological sample containing T cells is contacted ex vivo with antigen-presenting cells presenting one or more Mtb polypeptides comprising SEQ ID NO: 11; the sample is then contacted with capture monoclonal antibodies immobilized on solid supports for interferon-γ and subsequently with labeled antibodies specific to interferon-γ to detect complexes indicative of the presence of T cells recognizing the polypeptides, indicating Mtb infection.
The claims cover methods for detecting Mycobacterium tuberculosis infection based on the specific recognition of Mtb polypeptides, especially SEQ ID NO: 11, by T cells in biological samples using various immunoassays such as ELISPOT and cytokine detection, involving in vitro culture, ex vivo incubation with antigen-presenting cells, and detection of T cell activation markers.
Stated Advantages
Provides improved diagnostic accuracy for detecting tuberculosis infections by specifically identifying CD8+ T cell responses to defined Mtb polypeptides.
Enables differentiation between BCG vaccination and active or latent Mtb infection through specific T cell-based assays.
Allows detection of latent as well as active tuberculosis infections.
Documented Applications
Detection of Mycobacterium tuberculosis infection in subjects via in vitro assays that detect CD8+ T cells specific for Mtb polypeptides.
In vivo diagnostic skin tests measuring delayed type hypersensitivity reactions by intradermal administration of Mtb polypeptides.
Use of recovered polypeptides, polynucleotides, and T cell clone screening to identify immunodominant epitopes of Mtb for diagnostic purposes.
Monitoring progression of Mtb infection and evaluating effectiveness of therapeutic interventions by measuring the presence and frequency of T cells reactive to Mtb polypeptides.
Screening T cell responses against synthetic peptide libraries, including genomic peptide libraries representing Mtb genes, to identify novel diagnostic epitopes.
Use in animal models such as mice and guinea pigs for evaluating immune responses and protective efficacy of compositions containing Mtb polypeptides.
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