JP2011102299A5

JP2011102299A5 -

Info

Publication number: JP2011102299A5
Application number: JP2010262852A
Authority: JP
Filing date: 2010-11-25
Publication date: 2012-06-07
Anticipated expiration: 2020-07-13

Claims

1) a fragment having a length of at least 12 amino acid residues from the amino acid sequence set forth in SEQ ID NO: 17; or 2) having the same length as the sequence of 1) and having at least 95% sequence identity; The following characteristics:
i) Less than 20 μg / ml suspension in a suspension consisting of approximately 2 × 10 ⁵ T-lymphocytes isolated from the spleen of mice 28 days after infection with 5 × 10 ⁴ virulent mycobacteria Ability to induce an in vitro reaction during primary infection with virulent mycobacteria, as determined by adding oligopeptides at a concentration and IFN-γ in the supernatant recovered after 3 days is at least 1,500 pg / ml;
ii) About 1.0-2.5 × 10 ⁵ peripheral blood mononuclear cells (PBMC) or whole blood collected from TB patients or PPD positive individuals 0-6 months after diagnosis, suspended at 20 μg / ml Ability to induce an in vitro recall reaction as determined by adding oligopeptides at a concentration below the turbidity and IFN-γ in the supernatant recovered after 5 days is at least 500 pg / ml, preferably 1,000 pg / ml ;
iii) When the serum of TB patients is diluted 1:20 in PBS and incubated with oligopeptides at a high concentration of 20 μg / ml, an OD of at least 0.1 is obtained by ELISA or a reaction that can be visually confirmed by Western blot Ability to elicit a specific antibody response in TB patients as determined by causing;
iv) Suspensions of approximately 1.0-2.5 × 10 ⁵ peripheral blood mononuclear cells (PBMC) collected from clinically or semi-clinically toxic mycobacterial individuals with less than 20 μg / ml suspension By adding the oligopeptide at a concentration, the IFN-γ in the supernatant collected after 5 days is at least 500 pg / ml, preferably by not inducing the release of IFN-γ in individuals not infected with toxic mycobacteria The ability to elicit a positive in vitro response, as determined;
v) To the suspension of T cell lines from PPD positive individuals 1-5 × 10 ⁵ cells / ml, oligopeptide was added at a concentration of less than 20 μg / ml suspension and the supernatant recovered 3-5 days later Ability to elicit a positive in vitro response, as determined by having an IFN-γ in it of at least 500 pg / ml;
vi) The oligopeptide was added to a suspension of 1 to 5 × 10 ⁵ cells / ml of a T cell line from a PPD positive individual at a concentration of less than 20 μg / ml suspension, and the supernatant collected 3 to 5 days later Ability to elicit a positive in vitro response as determined by T cell proliferation with a stimulation index (SI) of at least 5 when assessed by the amount of IFN-γ in Calculated as [Average number per minute] / [Average number per minute without antigen]);
vii) Determined by showing a positive reaction at least 10 mm in diameter 72-96 hours after intradermal injection or topical application of up to 100 μg oligopeptide to individuals infected clinically or semiclinically with toxic mycobacteria Ability to induce a positive DTH response;
viii) When clinically or semiclinically toxic mycobacterial infected individuals show a positive reaction with a diameter of at least 5 mm after 72-96 hours when injected intradermally or topically with 100 μg of oligopeptide, preferably Comprises an oligopeptide having at least one ability to elicit a positive DTH response, determined by not inducing said response in individuals infected with virulent mycobacteria, said oligopeptide being recombinant in non-mycobacterial host cells An immunological composition produced by the method or synthesized by solid phase or liquid phase peptide synthesis methods.

The composition according to claim 1, wherein said oligopeptide fragment has a length of at least 20 amino acid residues.

The composition according to claim 1 or 2, wherein said oligopeptide fragment has a length of at least 24 amino acid residues.

4. The composition according to any one of claims 1 to 3, wherein the oligopeptide fragment has a length of at least 304 amino acid residues.

The composition according to any one of claims 1 to 4, wherein the oligopeptide fragment does not have any signal sequence.

Fusion comprising an oligopeptide as defined in any one of claims 1 to 4 and at least one fusion partner selected from the group consisting of ESAT-6, ESAT6 T cell epitope, CFP10 and CFP10 T cell epitope A composition according to any one of claims 1 to 4 comprising a protein.

7. A composition according to any one of claims 1 to 6, wherein the oligopeptide or fusion protein is lipidated to allow the oligopeptide or fusion protein to self-adjuvant.

8. A composition according to any one of claims 1 to 7, wherein the sequence identity of the oligopeptide is at least 98%.

An oligopeptide as defined in any one of claims 1 to 5, ESAT-6 (SEQ ID NO: 2), T cell epitope of ESAT6, CFP10 (SEQ ID NO: 1) and T cell epitope of CFP10 7. A composition according to claim 6 consisting of at least two different polypeptide fragments selected from the group consisting of.

The composition according to any one of claims 6 to 9, further comprising an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.

The carrier is selected from the group consisting of a polymer to which the oligopeptide binds by hydrophobic non-covalent interactions, a polymer to which the oligopeptide is covalently bonded, and a polypeptide; the vehicle is selected from the group consisting of a diluent and a suspending agent; And the adjuvant is dimethyl dioctadecyl ammonium bromide (DDA), Quil A, poly I: C, Freund's incomplete adjuvant, IFN-γ, IL-2, IL-12, monophosphoryl lipid A (MPL) and muramyl dipeptide ( A composition according to claim 10 selected from the group consisting of (MDP).

The composition according to any one of claims 1 to 11, which is in the form of a skin test reagent.

The composition according to any one of claims 1 to 11, which is a composition for detecting tuberculosis in animals including humans based on significant release of at least one cytokine by mononuclear cells.

13. A composition according to any one of claims 1 to 12 comprising detection means.

Preparation, synthesis or isolation of an oligopeptide as defined in any one of claims 1 to 5, and solubilization or dispersion of the oligopeptide in a suitable medium, optionally other M. tuberculosis antigens and / or Or a transformed cell comprising at least one replicable expression vector comprising a carrier, vehicle and / or adjuvant material, or comprising a nucleic acid sequence, the nucleic acid sequence according to any one of claims 1-5. 16. A method according to any of claims 8 to 15, comprising culturing transformed cells having a sequence encoding a defined oligopeptide and transferring the cells to a vaccine medium, optionally adding a carrier, vehicle and / or adjuvant material. A method for producing a composition by

Providing a blood sample derived from an animal or a human, comprising contacting the sample derived from an animal with the oligopeptide defined in any one of claims 1 to 5 or the fusion protein defined in claim 6; Past or present sensitization with bacteria belonging to the Mycobacterium tuberculosis group in animals or humans, where significant release of at least one cytokine into the extracellular phase by mononuclear cells in the sample indicates that the animal is sensitized How to detect.