Methods for detecting a Mycobacterium tuberculosis infection
Inventors
Lewinsohn, David M. • Lewinsohn, Deborah A.
Assignees
US Department of Veterans Affairs
Publication Number
US-8361707-B2
Publication Date
2013-01-29
Expiration Date
2027-03-14
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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. 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.
One third of the world's population harbors M. tuberculosis and is at risk for developing tuberculosis (TB). Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Diagnosis of tuberculosis is commonly achieved using a skin test that involves intradermal exposure to tuberculin PPD. However, the sensitivity and specificity of this test are not ideal; individuals vaccinated with BCG cannot be distinguished from infected individuals. Accordingly, there is a need in the art for improved diagnostic methods for detecting tuberculosis.
The methods disclosed include detecting CD8+ T cells and/or CD4+ T cells that specifically bind an Mtb polypeptide of interest, detecting a delayed type hypersensitivity reaction in a subject, and/or detecting specific Mtb polypeptides and polynucleotides. The Mycobacterium tuberculosis infection can be latent or active. The disclosed assays can be used individually or in combination. The Mtb polypeptides include specific amino acid sequences set forth as SEQ ID NOs: 1-12 or fragments thereof that specifically bind major histocompatibility complex (MHC) class I. T cells that specifically recognize these Mtb polypeptides are determined to detect infection. The invention also provides for reagents useful for detection of an Mtb infection.
Claims Coverage
The patent includes one independent claim that defines inventive features related to methods for detecting Mycobacterium tuberculosis infection by targeting T cells and Mtb polypeptides.
Detection of Mtb-specific T cell recognition in a biological sample
The method comprises contacting a biological sample comprising T cells derived from a subject with one or more isolated Mycobacterium polypeptides, wherein at least one polypeptide has a specific amino acid sequence (including SEQ ID NO: 8) and determining if the T cells specifically recognize the Mycobacterium polypeptide to detect Mtb infection in the subject.
Use of isolated Mycobacterium polypeptides with defined sequences
The isolated Mycobacterium polypeptide used in detection consists of the amino acid sequence set forth as SEQ ID NO: 8, or other specific SEQ ID NOs such as SEQ ID NOs: 1-7, 9, 11, or 12.
Focus on CD8+ T cells and cytokine secretion for detection
The T cells involved can be CD8+ T cells, and detection involves measuring secretion of cytokines such as interferon gamma (IFN-γ) from these CD8+ T cells, using antibodies specific to IFN-γ.
Biological sample types and cell culture conditions
The biological sample can be blood, isolated peripheral blood mononuclear cells, isolated mononuclear cells, or isolated T cells which can be cultured in vitro with the Mycobacterium polypeptide and/or with antigen presenting cells prior to detection.
The claims encompass methods detecting Mycobacterium tuberculosis infection through recognition of specific Mtb polypeptides by T cells in biological samples, focusing on CD8+ T cell responses and cytokine secretion, particularly IFN-γ, with defined Mtb polypeptides including SEQ ID NO: 8, and additional features relating to sample processing and in vitro culture for enhanced detection.
Stated Advantages
High frequency of CD8+ T cell responses specific for Mtb antigens, which can provide sensitive detection of infection.
Improved specificity of diagnosis distinguishing Mtb infection from BCG vaccination by targeting T cells that specifically recognize defined Mtb polypeptides.
Ability to detect both latent and active Mtb infections using in vitro and in vivo assays.
Documented Applications
Detection and diagnosis of Mycobacterium tuberculosis infection in subjects including latent and active tuberculosis.
Monitoring the progression of tuberculosis infection and the effectiveness of therapeutic protocols by measuring levels of Mtb polypeptides, polynucleotides, antibodies, or T cells specific to Mtb polypeptides.
Use in skin tests administering Mtb polypeptides intradermally to detect delayed type hypersensitivity reactions indicative of Mtb infection.
In vitro immunoassays detecting T cell activation, including ELISPOT assays measuring interferon gamma secretion from CD8+ T cells.
Use of reagents such as tetrameric MHC class I complexes incorporating Mtb polypeptides for detection of specific CD8+ T cells by fluorescence activated cell sorting (FACS).
Assays detecting antibodies reactive to Mtb polypeptides in biological samples using ELISA or rapid flow-through/strip test formats.
Molecular detection of Mtb polynucleotides encoding Mtb polypeptides by amplification and hybridization techniques such as PCR and real-time quantitative PCR.
Animal models including mouse and guinea pig models for evaluating compositions for vaccination or treatment by monitoring T cell responses to Mtb polypeptides.
Interested in licensing this patent?