# US 9,040,233 B2 · Methods for detecting a Mycobacterium tuberculosis infection > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,233 B2 - Document ID: US9040233B2 - Status: Granted - Kind: B2 - Application number: 13/719,124 - Filed: 2012-12-18 - Published: 2013-04-25 - Granted: 2015-05-26 - Earliest claimed: 2011-09-23 - Assignees: Oregon Health & Science University; The United States of America, DBA The Department of Veteran Affairs - Applicants: Oregon Health & Science University; The Government of the United States of America, dba The Department of Veterans Affairs - Inventors: David M. Lewinsohn; Deborah A. Lewinsohn - Web page: https://patentgrep.com/patent/US9040233B2 ## CPC - G01N 33/5695: {Mycobacteria} - A61K 39/04: Mycobacterium, e.g. Mycobacterium tuberculosis - A61P 31/04: Antibacterial agents - A61P 31/06: for tuberculosis - A61P 37/04: Immunostimulants - C07K 14/35: from Mycobacteriaceae (F) - G01N 33/505: {involving T-cells} - G01N 33/6893: {related to diseases not provided for elsewhere} - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - A61K 2039/53: {DNA (RNA) vaccination} - G01N 2333/35: from Mycobacteriaceae (F) - G01N 2469/10: Detection of antigens from microorganism in sample from host - G01N 2800/26: Infectious diseases, e.g. generalised sepsis - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## 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. ## Claims ### Claim 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: 11; 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. ### Claim 2 (depends on 1) The method of claim 1, wherein one of the isolated Mycobacterium polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 11. ### Claim 3 (depends on 1) The method of claim 1, wherein the T cells are CD8+ T cells. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) The method of claim 1, wherein the biological sample is blood, isolated peripheral blood mononuclear cells, or isolated mononuclear cells. ### Claim 6 (depends on 1) The method of claim 1, wherein the biological sample is isolated T cells. ### Claim 7 (depends on 6) 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: 11. ### Claim 8 (depends on 6) The method of claim 6, wherein the T cells are cultured in vitro with antigen presenting cells. ### Claim 9 (depends on 7) 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: 11. ### Claim 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: 11; 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. ### Claim 11 (depends on 10) The method of claim 10, wherein the biological sample is blood, isolated peripheral blood mononuclear cells, or isolated mononuclear cells. ### Claim 12 (depends on 10) The method of claim 10, wherein the labeled monoclonal antibody is labeled with an enzyme, a fluorescent marker, biotin or a radioactive marker. ### Claim 13 (depends on 12) 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. ### Claim 14 (depends on 10) 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: 11. ### Claim 15 A method for detecting Mycobacterium tuberculosis in a subject, comprising: incubating a biological sample comprising 105 to 107 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: 11 and is present at a concentration of 10−1 to 103 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). ### Claim 16 (depends on 15) The method of claim 15, wherein one of the isolated Mycobacterium polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 11. ### Claim 17 (depends on 15) The method of claim 15, wherein the T cells are CD8+ T cells. ### Claim 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: 11 and is present at a concentration of 10−1 to 103 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). ### Claim 19 (depends on 17) The method of claim 17, wherein one of the isolated Mycobacterium polypeptides consists of the amino acid sequence set forth as SEQ ID NO: 11.