Methods for detecting a Mycobacterium tuberculosis infection
Inventors
Lewinsohn, David M. • Lewinsohn, Deborah A.
Assignees
US Department of Veterans Affairs
Publication Number
US-8053181-B2
Publication Date
2011-11-08
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
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. These methods encompass in vitro assays for detecting CD8+ T cells in a biological sample and in vivo assays that detect a delayed type hypersensitivity reaction. Additionally, methods for detecting Mtb polypeptides and polynucleotides are described. Reagents useful for detecting an Mtb infection are also disclosed.
Mycobacteria are intracellular bacteria responsible for diseases including tuberculosis, which affects one third of the world’s population. Diagnosing tuberculosis currently relies on a skin test with tuberculin purified protein derivative (PPD), but this test has limitations in sensitivity and specificity and cannot distinguish between BCG vaccinated and infected individuals. There is an increasing need for improved, accurate, and early diagnostic methods for tuberculosis.
The disclosed methods address this need by identifying Mycobacterium tuberculosis infections through detection of specific T cell responses, including CD8+ and CD4+ T cells that recognize defined Mtb polypeptides. The methods also enable detecting delayed hypersensitivity reactions via skin tests using defined Mtb polypeptides and involve detecting Mycobacterium polypeptides and encoding polynucleotides in biological samples. These assays can be used alone or in combination, applicable to detecting latent or active Mtb infections.
Claims Coverage
The patent claims include two independent methods for detecting Mycobacterium tuberculosis infection involving detection of immune cells recognizing a specific Mtb polypeptide and detection of the polypeptide or its encoding nucleic acid in biological samples. The main inventive features are focused on these detection approaches using defined polypeptide sequences and associated detection techniques.
Detection of T cells recognizing a specific Mtb polypeptide in a biological sample
A method comprising contacting a biological sample from a subject containing T cells with one or more isolated Mycobacterium polypeptides, where one polypeptide is the amino acid sequence of SEQ ID NO: 10, and determining if the T cells specifically recognize this sequence, thereby detecting Mtb infection.
Use of CD8+ T cells secreting cytokines as detection marker
The detection of CD8+ T cells specifically recognizing the Mtb polypeptide is performed by measuring cytokine secretion, in particular interferon-gamma (IFN-γ), indicating recognition of the antigen.
Detection of Mycobacterium polypeptide or encoding polynucleotide in a biological sample
A method comprising detecting the presence of an Mtb polypeptide of SEQ ID NO: 10 or its encoding polynucleotide, including mRNA, in a biological sample from the subject to indicate Mtb infection.
Use of antibodies specific to Mtb polypeptide for detection
Detection of the Mtb polypeptide in a biological sample is conducted by using an antibody that specifically binds to the Mtb polypeptide SEQ ID NO: 10.
In vitro culturing of T cells with Mtb polypeptide and antigen-presenting cells
Isolated T cells from the subject are cultured in vitro, optionally with antigen-presenting cells, prior to or during incubation with the Mtb polypeptide to detect specific recognition.
Administration of the Mtb polypeptide to the subject for in vivo detection
The Mtb polypeptide is administered to the subject, and the presence of T cells recognizing the polypeptide in vivo is determined by measuring a delayed type hypersensitivity or T cell response.
The claims collectively cover methods for detecting Mtb infection by identifying T cells specifically recognizing a defined Mtb polypeptide, particularly SEQ ID NO: 10, measuring cytokine secretion such as IFN-γ, detecting the Mtb polypeptide or its encoding nucleic acid in biological samples, and using in vitro culturing or in vivo administration to establish infection presence.
Stated Advantages
The disclosed methods provide improved accuracy and specificity for detecting Mycobacterium tuberculosis infection compared to existing tests like the tuberculin skin test.
The methods enable detection of latent and active tuberculosis infections by identifying specific CD8+ and CD4+ T cell responses to defined Mtb polypeptides.
The use of defined Mtb polypeptides in assays allows differentiation of infected individuals from those vaccinated with BCG, overcoming limitations of current diagnostics.
The invention provides reagents such as peptide-MHC tetramers for sensitive detection and quantification of Mtb-specific T cells in biological samples.
Documented Applications
In vitro assays for detecting CD8+ T cells specific to Mtb polypeptides in biological samples such as blood, isolated peripheral blood mononuclear cells, sputum, lymph node tissue, lung tissue, or isolated T cells.
In vivo assays involving administration of Mtb polypeptides to a subject to induce and detect a delayed type hypersensitivity skin reaction indicative of Mtb infection.
Detection of Mtb polypeptides or polynucleotides including mRNA in biological samples using antibodies or nucleic acid amplification techniques such as PCR, Northern blot, or dot blot assays.
Use of Mtb-specific reagents including tetrameric MHC class I/peptide complexes to identify and quantify antigen-specific CD8+ T cells by flow cytometry.
Monitoring progression of tuberculosis infection or effectiveness of therapy by measuring changes over time in levels of Mtb polypeptides, polynucleotides, or antigen-specific T cell populations in a subject.
Application of the diagnostic methods in various animal models including mouse and guinea pig tuberculosis infection models for vaccine development and evaluation of protective immunity.
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