Use of amino acid sequences from mycobacterium tuberculosis or corresponding nucleic acids for diagnosis and prevention of tubercular infection, diagnostic kit and vaccine therefrom
The present invention refers to the use of gene sequences or portions thereof characterized in that the same belong to the classes of in vitro and ex vivo induced, repressed or conserved genes in Mycobacterium tuberculosis currently infected human macrophages and to corresponding peptides or consensus peptides or proteins for the preparation of specific bio-markers for the diagnosis and prevention of active or latent disease.
1. A method for detecting a Mycobacterium tuberculosis infection in a test subject, comprising:
(A) incubating in vitro (i) a blood sample or a lavage sample that comprises lymphocytes from the test subject, with (ii) ESAT6 protein and CFP10 protein, and (iii) at least one of:
(a) at least 6 peptides that each comprise at least one Mycobacterium tuberculosis T-cell epitope, said peptides being selected from TAWITAWPGLMV (SEQ ID NO:24), ELMARAAVLGSAH (SEQ ID NO:21), RPVRRVLLFWPSSGPAP (SEQ ID NO:70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO:71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO:22), LAWITAWPGLMV (SEQ ID NO:85), GEIIFISGRLNG (SEQ ID NO:86) and SALLRRLSTCPPES (SEQ ID NO:87),
(b) at least 6 peptides that each comprise at least one Mycobacterium tuberculosis T-cell epitope, said peptides being selected from ELMARAAVLGSAH (SEQ ID NO:21), RPVRRVLLFWPSSGPAP (SEQ ID NO:70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO:71), AVIVRSELLTQYL (SEQ ID NO:22), TAWITAWPGLMV (SEQ ID NO:24) and GEIIFISGRLNGaa (SEQ ID NO:13), and
(c) at least 6 peptides that each comprise at least one Mycobacterium tuberculosis T-cell epitope, said peptides being selected from ELMARAAVLGSAH (SEQ ID NO:21), RPVRRVLLFWPSSGPAP (SEQ ID NO:70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO:71), AVIVRSELLTQYL (SEQ ID NO:22), LAWITAWPGLMV (SEQ ID NO:85) and GEIIFISGRLNG (SEQ ID NO:86),
wherein said step of incubating is for a time and under conditions sufficient for:
(1) recognition, by one or more of a first plurality of T cell receptors (TCR) of T cells that are present in said lymphocytes, of one or more of said at least 6 peptides of (a), (b), or (c) to stimulate the lymphocytes to produce an effector molecule, and
(2) recognition, by one or more of a second plurality of T cell receptors (TCR) of T cells that are present in said lymphocytes, of one or more of said ESAT6 and CFP10 proteins to stimulate the lymphocytes to produce the effector molecule; and
(B) detecting a test level of the effector molecule that is produced by lymphocytes stimulated according to step (A), wherein the detected test level of the effector molecule is greater than a detectable effector molecule level that can be produced by said lymphocytes incubated only with said ESAT6 and CFP10 proteins,
wherein if the test level of the effector molecule that is detected is higher than a control level of the effector molecule that is detected following incubation of control lymphocytes from a healthy control subject, then the test level indicates that the test subject is infected with, or has been previously exposed to, Mycobacterium tuberculosis , and wherein the effector molecule is selected from (i) a cytokine or an interleukin the test level of which is indicative of the test subject's capacity to mount a cell-mediated immune response, or (b) IFN-γ or TNF-α.
2. The method of claim 1 in which incubating further comprises incubating with TB7.7 protein for a time and under conditions sufficient for recognition, by one or more of said second plurality of T cell receptors (TCR) of T cells that are present in said lymphocytes, of said TB7.7 protein to stimulate the lymphocytes to produce the effector molecule, further wherein the detected test level of the effector molecule is greater than a detectable effector molecule level that can be produced by lymphocytes incubated only with said ESAT6, CFP10 and TB7.7 proteins.
3. The method of claim 1 wherein the effector molecule is selected from interferon-γ, TNF-α, and an interleukin.
4. The method of claim 1 wherein the effector molecule is interferon-γ.