Antigenic tripeptides derived from Mycobacterium avium subsp. paratuberculosis s-type strains, derivatives and uses thereof
Inventors
Bannantine, John P. • Etienne, Gilles • Bay, Sylvie • Biet, Franck
Assignees
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement • Institut Pasteur • US Department of Agriculture USDA
Publication Number
US-11981750-B2
Publication Date
2024-05-14
Expiration Date
2037-12-20
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Abstract
The present invention is directed to an isolated synthetic tripeptide of formula H-D-Phe-N-Methyl-L-Val-L-Ala-OMe (SEQ ID NO:1), or a derivative thereof, and to the corresponding lipotripeptides, which are specific to Mycobacterium avium subsp. paratuberculosis (Map) S-type strain, as well as derivatives and conjugates thereof. The invention also concerns the use of these antigens in different methods and tests for detecting Map infection, especially by detecting humoral response and cell mediated response of infected animals. The invention is also directed to a genetic signature of Map and a mass spectrometry and NMR spectroscopy signature of Map presence or infection.
Core Innovation
The present invention is directed to isolated synthetic tripeptides of formula H-D-Phe-N-Methyl-L-Val-L-Ala-OMe (SEQ ID NO:1) or derivatives thereof, and to corresponding lipotripeptides specific to Mycobacterium avium subsp. paratuberculosis (Map) S-type strains, as well as derivatives and conjugates thereof. The invention also concerns the use of these antigens in diagnostic methods for detecting Map infection, particularly by detecting humoral and cell-mediated immune responses of infected animals. Additionally, the invention relates to a genetic signature and mass spectrometry and NMR spectroscopy signatures indicating Map presence or infection.
The problem being solved arises from the difficulty to accurately diagnose Johne's disease caused by Map, especially in early stages. Existing diagnostics lack specificity and sensitivity, in particular for discriminating between S-type and C-type Map strains and from other mycobacteria such as M. avium subsp. avium and M. bovis. The lipopentapeptide L5P previously identified as a Map antigen is specific to C-type strains and absent from S-type strains, limiting diagnostic options for the latter. There is a need for improved, simple, rapid, and specific diagnostic tests suitable for use by veterinarians or producers without expensive equipment, which limit undetermined results and allow discrimination between Map strains and other mycobacteria.
The inventors discovered that S-type Map strains produce a distinct lipotripeptide (L3P) composed of the tripeptide H-D-Phe-N-Methyl-L-Val-L-Ala-OMe attached to a lipid moiety, differing from the lipopentapeptide produced by C-type strains. This tripeptide and its conjugates form a specific antigenic signature for S-type Map, recognized by antibodies from infected animals. The invention discloses synthetic analogues, including hydrosoluble variants, for use in diagnostic methods detecting humoral and cell-mediated responses. Furthermore, genetic methods exploiting a 6.3 kb deletion in the mps1 gene unique to S-type strains enable genetic discrimination between Map S-type and C-type.
Claims Coverage
The claims include multiple independent claims covering genetic discrimination methods of Map S-type and C-type strains, involving detecting a 6.3 kb deletion in the mps1 gene, and amplification with specific primers.
Genetic discrimination of Map S-type versus C-type by detecting a 6.3 kb deletion in the mps1 gene
A method comprising detecting a 6.3 kb deletion in the mps1 gene of Map S-type with respect to Map C-type, for genetic discrimination of the strain type in a sample.
Method for genetic characterization of Map S-type via PCR amplification with specific primers
A method involving PCR amplification of genomic DNA of tested mycobacterium using forward primer P1 (SEQ ID NO:10) and reverse primer P3 (SEQ ID NO:12) to detect an amplified product of about 1112 bases indicative of Map S-type strain presence.
Genetic discrimination between Map S-type and C-type by primer-based PCR
A method involving PCR amplification with primers P1 (SEQ ID NO:10), P2 (SEQ ID NO:11), and P3 (SEQ ID NO:12) where the length of amplified products indicates the strain: approximately 1112 bases for S-type and approximately 356 bases for C-type strains.
The claims cover the use of genetic markers specific for Map S-type involving a distinct deletion in the mps1 gene and primer-based PCR amplification methods to discriminate between S-type and C-type Map strains in biological samples.
Stated Advantages
The invention provides a highly specific antigen for Map S-type strains allowing early and accurate detection of infection.
Hydrosoluble analogues of lipopentapeptide and lipotripeptide enable diagnostic tests that do not require organic solvents, facilitating use by non-specialists.
Methods enable discrimination between Map infection and infections by related mycobacteria such as M. avium subsp. avium and M. bovis.
The antigenic compounds elicit both humoral and cell-mediated immune responses, enabling improved diagnostic sensitivity.
Documented Applications
Use of synthetic tripeptide and lipotripeptide antigens for serological (antibody-based) and cell-mediated immune response diagnostic tests to detect Map infection, especially Map S-type, in animals such as sheep, goats, and cattle.
Genetic methods for strain typing of Map in biological samples via PCR amplification using specific primers, allowing discrimination between S-type and C-type strains.
Use of mass spectrometry (e.g. MALDI-TOF) and NMR spectroscopy signatures of lipotripeptides for detection of Map S-type presence in biological samples.
Immunogenic compositions comprising the tripeptides or conjugates for vaccination against Map S-type infection in animals.
Diagnostic kits incorporating the antigens and reagents for detecting antigen-antibody complexes to diagnose Map infection.
Investigation of human Crohn's disease patients for Map infection using the disclosed antigens and methods.
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