Synthetic antigenic peptides and lipopeptides derived from
The current invention relates to the diagnosis and treatment of diseases resulting from infections by Mycobacterium avium subsp. paratuberculosis . In particular the invention relates to the use of an antigen selected among (a) a synthetic peptide 5P having the following formula: DPhe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 1); (b) a lipopeptide L5P consisting of the synthetic peptide a) wherein the N-terminal phenylalanine residue is N-acylated with an eicosanoic acid acyl chain; (c) a variant of peptide a) or lipopeptide b) able to react with anti- Mycobacterium paratuberculosis antibodies; for in vitro detection or quantification of specific anti- Mycobacterium paratuberculosis antibodies in a biological sample.
1. A method of in vitro detection of specific anti- Mycobacterium paratuberculosis antibody comprising in a first step contacting a biological sample with an antigen for an amount of time sufficient to form an antigen-antibody complex, wherein the antigen is selected from the group consisting of:
(a) a synthetic peptide 5P having the following formula:
DPhe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 1);
(b) a lipopeptide L5P consisting of the synthetic peptide a) wherein the N-terminal phenylalanine residue is N-acylated with an eicosanoic acid acyl chain; and
(c) a variant of peptide (a) or lipopeptide (b) which reacts with anti- Mycobacterium paratuberculosis antibodies, and in a second step detecting the formation of the antigen-antibody complex in said biological sample, wherein the presence of the antigen-antibody complex indicates the presence of the anti- Mycobacterium paratuberculosis antibody.
2. The method of claim 1 , wherein said variant is selected from the group consisting of:
Phe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 2);
DPhe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 3);
Phe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 4);
DPhe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 5);
DPhe-Val-Ile-Phe-Ala (SEQ ID NO: 6);
Phe-Val-Ile-Phe-Ala (SEQ ID NO: 7);
Phe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 8)
and their derivatives resulting from N-acylation of the N-terminal phenylalanine residue with a C1 to C30 acyl group.
3. A method of evaluating in vitro the T-cell immune response directed against Mycobacterium paratuberculosis in a subject comprising the steps of contacting a biological sample containing T cells isolated from the subject, with an antigen as defined in claim 1 , and detecting cytokine expression by activated T-cells in the biological sample, wherein cytokine expression indicates the presence of a T cell immune response in the subject.
4. The method of claim 1 , where in the antigen-antibody complex is detected using a technique selected from the group consisting of ELISA, radioimmunoassay, electrophoresis, immunofluorescence and western blot.
5. The method of claim 2 , wherein said variant is selected from the group consisting of:
Phe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 2);
DPhe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 3);
Phe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 4);
DPhe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 5);
DPhe-Val-Ile-Phe-Ala (SEQ ID NO: 6);
Phe-Val-Ile-Phe-Ala (SEQ ID NO: 7); and
Phe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 8).
6. The method of claim 2 , wherein said variant is selected from the group consisting of:
Phe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 2);
DPhe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 3);
Phe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 4);
DPhe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 5);
DPhe-Val-Ile-Phe-Ala (SEQ ID NO: 6);
Phe-Val-Ile-Phe-Ala (SEQ ID NO: 7); and
Phe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 8),
wherein the N-terminal phenylalanine residue is N-acylated with a C1 to C30 acyl group.
7. The method of claim of claim 3 , wherein the cyokine expression is detected using ELISPOT.
8. A composition comprising an antigen selected from the group consisting of:
(a) a synthetic peptide 5P having the following formula:
DPhe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 1);
(b) a lipopeptide L5P consisting of the synthetic peptide a) wherein the N-terminal phenylalanine residue is N-acylated with an eicosanoic acid acyl chain; and
(c) a variant of peptide (a) or lipopeptide (b) which reacts with anti- Mycobacterium paratuberculosis antibodies;
wherein the antigen is present in an amount sufficient to form an antigen-antibody complex with an anti- Mycobacterium paratuberculosis antibody.
9. The composition of claim 8 , wherein said variant is selected among the group consisting of:
Phe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 2);
DPhe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 3);
Phe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 4);
DPhe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 5);
DPhe-Val-Ile-Phe-Ala (SEQ ID NO: 6);
Phe-Val-Ile-Phe-Ala (SEQ ID NO: 7);
Phe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 8)
and their derivatives resulting from N-acylation of the N-terminal phenylalanine residue with a C1 to C30 acyl group.
10. The composition of claim 9 , wherein said variant is selected among the group consisting of:
Phe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 2);
DPhe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 3);
Phe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 4);
DPhe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 5);
DPhe-Val-Ile-Phe-Ala (SEQ ID NO: 6);
Phe-Val-Ile-Phe-Ala (SEQ ID NO: 7); and
Phe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 8).
11. The composition of claim 9 , wherein said variant is selected among the group consisting of:
Phe-NMeVal-Ile-Phe-Ala-OMe (SEQ ID NO: 2);
DPhe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 3);
Phe-Val-Ile-Phe-Ala-OMe (SEQ ID NO: 4);
DPhe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 5);
DPhe-Val-Ile-Phe-Ala (SEQ ID NO: 6);
Phe-Val-Ile-Phe-Ala (SEQ ID NO: 7); and
Phe-NMeVal-Ile-Phe-Ala (SEQ ID NO: 8),
wherein the N-terminal phenylalanine residue is N-acylated with a C1 to C30 acyl group.
12. The composition of claim 8 wherein the composition is an immunogenic composition.