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 la Recherche Agronomique INRA • Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement • Institut Pasteur • US Department of Agriculture USDA
Publication Number
US-11498942-B2
Publication Date
2022-11-15
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 invention relates to isolated synthetic tripeptides of formula H-D-Phe-N-Methyl-L-Val-L-Ala-OMe (SEQ ID NO:1), or derivatives thereof, which are specific antigens of Mycobacterium avium subsp. paratuberculosis (Map) S-type strains. These tripeptides, as well as their lipotripeptide conjugates and variants, serve as molecular signatures for detecting Map S-type infections by eliciting specific humoral and cell-mediated immune responses in infected animals.
The background recognizes the difficulty of early and accurate diagnosis of Johne's disease caused by Map, especially due to the pathogen's latency, the similarity between Map and other Mycobacterium avium subspecies antigenically, and the lack of specific antigenic markers for S-type strains. Previous antigen-based tests suffer from cross-reactivity and lack sensitivity, particularly for S-type isolates, which hinders disease control. The problem solved is the identification and synthetic preparation of a specific antigenic tripeptide unique to Map S-type strains that can be used in diagnostic tests and immunogenic compositions, enabling specific, sensitive, and early detection of Map S-type infections, which was not previously achievable.
Claims Coverage
The patent contains multiple independent claims focused on isolated synthetic tripeptides specific to Map S-type strains, compositions comprising these tripeptides, diagnostic kits using these antigens, and diagnostic and immune response evaluation methods using these antigens. The main inventive features emphasize the specific synthetic tripeptides, their chemical variants and conjugates, and methods employing them for detecting and diagnosing Map S-type infection.
Isolated synthetic tripeptides specific to Map S-type
An isolated synthetic tripeptide chosen from a defined group of eight formula variants of H-D-Phe-N-Methyl-L-Val-L-Ala-OMe and related derivatives (SEQ ID NOs: 1-8), representing the peptidyl moiety specific to Map S-type strains.
Composition comprising Map S-type specific synthetic tripeptides
A composition comprising an antigen specific to Map S-type chosen from isolated synthetic tripeptides as defined above, or such tripeptides N-acylated at the N-terminal Phenylalanine with a C1 to C30 acyl moiety that is not a fatty acid moiety.
Diagnostic kit for Map S-type infection
A diagnostic kit for detecting Map S-type infection containing an antigen as above (isolated synthetic tripeptide of SEQ ID NOs:1-8 or their N-acylated derivatives) and reagents for detecting antigen-antibody complexes.
Antigenic composition combining S-type tripeptide and C-type pentapeptide conjugates
An antigenic composition comprising (A) the isolated synthetic tripeptides or their N-acylated derivatives specific to Map S-type and (B) a hydrosoluble pentapeptide conjugate with the pentapeptide core H-Phe-Val-Ile-Phe-Ala-OH (SEQ ID NO: 13) modified by amidation and polyethylene glycol moieties, to provide diagnostic or immunogenic functionality covering Map S- and C-types.
Method for in vitro detection of anti-Map antibodies using specific tripeptides
A method for detecting specific anti-Map antibodies in a sample by contacting it with an antigen chosen from the isolated synthetic tripeptides (SEQ ID NOs:1-8) or their N-acylated derivatives, followed by detecting antigen-antibody complexes indicative of infection.
Method for diagnosing Map infection using synthetic tripeptides
A method for diagnosing Map infection in a subject by contacting a sample with antigens as above and detecting antigen-antibody complexes, indicative of infection or infection history in subjects not vaccinated against Map.
Method for detecting humoral immune response against Map
A method for detecting humoral immune response directed against Map using isolated synthetic tripeptides or their N-acylated derivatives specific to Map S-type, by forming and detecting antigen-antibody complexes.
Method for evaluating in vitro cell-mediated immune response against Map
A method for evaluating cell-mediated immune response directed against Map by contacting a biological sample with the isolated synthetic tripeptides or their N-acylated derivatives and detecting cytokine expression (preferably IFNγ or IL-10) or lymphoproliferation.
The claims cover the isolated synthetic tripeptides specific to Map S-type strains and their chemical variants, compositions and kits comprising these antigens, and multiple diagnostic and immune response evaluation methods employing these antigens. These inventive features provide sensitive and specific detection, differentiation, and vaccination means for Map S-type, also including hydrosoluble analogues for C-type strains to achieve broader diagnostic and therapeutic coverage.
Stated Advantages
The antigens allow specific detection of Map S-type infections with high diagnostic value, overcoming limitations of existing tests that lack specificity for S-type strains.
The synthetic tripeptides and their hydrosoluble derivatives enable the development of simple, rapid, and noninvasive diagnostic tests that can be performed without expensive laboratory equipment.
The antigens allow early detection of infection, including subclinical stages, by detecting humoral and cell-mediated immune responses.
The hydrosoluble analogues improve handling and test performance compared to natural hydrophobic lipopeptides, facilitating field use.
The antigens enable differentiation between Map infection and infections by other mycobacteria such as M. bovis, reducing false positives and improving disease control.
The compositions can be used for immunization or vaccination against Map S-type infection, potentially preventing disease and limiting economic losses.
Documented Applications
Diagnosis of Mycobacterium avium subsp. paratuberculosis (Map) S-type infection in animals, especially sheep and goats, through antibody and cell-mediated immune response detection.
Early and specific detection of Map infection in subjects, including subclinical stage identification, useful for disease management and control in livestock.
Discrimination between Map S-type and C-type infections and differentiation from other mycobacterial infections such as M. bovis.
Use in diagnostic kits for veterinary use applicable by veterinarians or animal producers without specialized equipment.
Use in immunogenic compositions or vaccines to induce protective immune responses against Map S-type infection in susceptible animals.
Detection and diagnostic studies in human subjects, particularly relating to the suspected association between Map and Crohn's Disease.
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