Methods for detecting a mycobacterium tuberculosis infection

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.

1 . A method for detecting  Mycobacterium tuberculosis  in a subject, comprising:
 contacting a biological sample from the subject, wherein the biological sample comprises T cells, with one or more-isolated  Mycobacterium  polypeptides in vitro, wherein one of the isolated  Mycobacterium  polypeptides comprises the amino acid sequence set forth as SEQ ID NO: 7; and   performing an enzyme-linked immunospot assay that detects interferon-γ in order to determine if the T cells specifically recognize the  Mycobacterium  polypeptide, wherein a positive enzyme-linked immunospot assay that detects interferon-γ in the biological sample determines the presence of T cells that specifically recognize the  Mycobacterium  polypeptide and detects  Mycobacterium tuberculosis  in the subject.   
     
     
         2 . The method of  claim 1 , wherein one of the isolated  Mycobacterium  polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 7. 
     
     
         3 . The method of  claim 1 , wherein the T cells are CD8+ T cells. 
     
     
         4 . The method of  claim 1 , further comprising measuring the size of the T cells, the proliferation of the T cells, or a cell surface marker of the T cells. 
     
     
         5 . The method of  claim 1 , wherein the biological sample is blood, isolated peripheral blood mononuclear cells, or isolated mononuclear cells. 
     
     
         6 . The method of  claim 1 , wherein the biological sample is isolated T cells. 
     
     
         7 . The method of  claim 6 , wherein the isolated T cells are cultured in vitro prior to contacting the biological sample with one or more-isolated  Mycobacterium  polypeptides, wherein one of the isolated  Mycobacterium  polypeptides comprises the amino acid sequence set forth as SEQ ID NO: 7. 
     
     
         8 . The method of  claim 6 , wherein the T cells are cultured in vitro with antigen presenting cells. 
     
     
         9 . The method of  claim 7 , wherein the one of the isolated  Mycobacterium  polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 7. 
     
     
         10 . A method for detecting a  Mycobacterium tuberculosis  infection in a subject, comprising:
 contacting ex vivo a biological sample from the subject, wherein the biological sample comprises T cells, with antigen presenting cells presenting one or more  Mycobacterium  polypeptides, wherein the amino acid sequence of one of the one or more  Mycobacterium  polypeptides comprises the amino acid sequence set forth as SEQ ID NO: 7; and   contacting the sample with a capture monoclonal antibody that specifically binds interferon γ, wherein the capture monoclonal antibody is immobilized on a solid support, to form a complex; and   contacting the sample with an additional labeled antibody that specifically binds interferon γ to detect the presence of the complex, wherein the presence of the labeled antibody bound to the complex indicates the presence of T cells that specifically recognize one of the  Mycobacterium  polypeptides and detects the  Mycobacterium tuberculosis  infection in the subject. 25   
     
     
         11 . The method of  claim 10 , wherein the biological sample is blood, isolated peripheral blood mononuclear cells, or isolated mononuclear cells. 
     
     
         12 . The method of  claim 10 , wherein the labeled monoclonal antibody is labeled with an enzyme, a fluorescent marker, biotin or a radioactive marker. 
     
     
         13 . The method of  claim 12 , wherein the labeled monoclonal antibody is labeled with biotin, and the method further comprises contacting the sample with streptavidin conjugated to an enzyme, a fluorescent marker or a radioactive marker. 
     
     
         14 . The method of  claim 10 , wherein the one of the isolated  Mycobacterium  polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 7. 
     
     
         15 . A method for detecting  Mycobacterium tuberculosis  in a subject, comprising:
 incubating a biological sample comprising 10 5  to 10 7  peripheral blood mononuclear cells isolated from the subject in vitro with one or more-isolated  Mycobacterium  polypeptides in a culture wherein one of the isolated  Mycobacterium  polypeptides comprises the amino acid sequence set forth as SEQ ID NO: 7 and is present at a concentration of 10 −1  to 10 3  μg per ml of the culture, and wherein the peripheral blood mononuclear cells are incubated with the polypeptide for about 4 to about 24 hours; and   performing an assay in order to determine if T cells in the biological sample specifically recognize the  Mycobacterium  polypeptide, wherein the presence of T cells that specifically recognize the  Mycobacterium  polypeptide and detects  Mycobacterium tuberculosis  in the subject, wherein the assay comprises one or more of a) determining if the polypeptide binds T cells; b) evaluating cytolytic activity of the T cells; c) evaluating proliferation of the T cells, d) detecting the secretion of interferon gamma, interleukin-2 or tumor necrosis factor alpha, or e) any combination of a)-d).   
     
     
         16 . The method of  claim 15 , wherein one of the isolated  Mycobacterium  polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 7. 
     
     
         17 . The method of  claim 16 , wherein the T cells are CD8+ T cells. 
     
     
         18 . A method for detecting  Mycobacterium tuberculosis  in a subject, comprising:
 isolating CD8+ T cells from a biological sample from the subject;   incubating the CD8+ T cells isolated from the subject in vitro with antigen presenting cells and with one or more-isolated  Mycobacterium  polypeptides in a culture, wherein one of the isolated  Mycobacterium  polypeptides comprises the amino acid sequence set forth as SEQ ID NO: 7 and is present at a concentration of 10 −1  to 10 3  μg per ml of the culture, and wherein the CD8+ T cells are incubated with the polypeptide for about 4 to about 24 hours; and   performing an assay in order to determine if T cells in the biological sample specifically recognize the  Mycobacterium  polypeptide, wherein the presence of T cells that specifically recognize the  Mycobacterium  polypeptide and detects  Mycobacterium tuberculosis  in the subject, wherein the assay comprises one or more of a) determining if the polypeptide binds T cells; b) evaluating cytolytic activity of the T cells; c) evaluating proliferation of the T cells, d) detecting the secretion of interferon gamma, interleukin-2 or tumor necrosis factor alpha, or e) any combination of a)-d).   
     
     
         19 . The method of  claim 18 , wherein one of the isolated  Mycobacterium  polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 7.