Engineering synthetic lipopeptide antigen for specific detection of Mycobacterium avium subsp. paratuberculosis infection

Authors: BAY, SYLVIE - Institut Pasteur - France; BEGG, DOUGLAS - University Of Sydney; GANNEAU, CHRISTELLE - Institut Pasteur - France; BRANGER, MAXIME - Universite De Tours; COCHARD, THIERRY - Universite De Tours; Bannantine, John; KOHLER, HEIKE - Friedrich-Loeffler-institut; MOYEN, JEAN LOUIS - Universite De Tours; WHITTINGTON, RICHARD - University Of Sydney; BIET, FRANCK - Universite De Tours

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2021
Publication Date: 4/23/2021
Citation: Bay, S., Begg, D., Ganneau, C., Branger, M., Cochard, T., Bannantine, J.P., Kohler, H., Moyen, J., Whittington, R.J., Biet, F. 2021. Engineering synthetic lipopeptide antigen for specific detection of Mycobacterium avium subsp. paratuberculosis infection. Frontiers in Veterinary Science. 8. https://doi.org/10.3389/fvets.2021.637841.
DOI: https://doi.org/10.3389/fvets.2021.637841

Interpretive Summary: This paper examines a unique diagnostic molecule that can be used for Johne's disease detection. In this work we chemically synthesized a lipid molecule that is attached to a 5-amino acid peptide. This molecule, termed a lipopentapeptide, is found naturally in the cell wall of Mycobacterium avium subspecies paratuberculosis and is antigenic. The goal was to synthesize enough of the L5P molecule to analyze if it can distinguish healthy cows from cows with Johne's disease. The result was that it can distinguish healthy from disease cows, but not as well as the commercially available test from IDEXX. Also, because the lipid molecule is not soluble in water, we synthesized a derivative of L5P that is water soluble. We found this derivative to be just as antigenic as the native form. A patent application on the antigenic lipid molecule described in this work was published on January 23, 2020 (PCT/EP2017/083924).

Technical Abstract: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce GPL on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18–C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide compound in large quantities for immunological studies. Various studies described the immune response directed against this purified or synthetic LP to confirm its immunogenic potential and capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analogue were synthesized on solid phase using Fmoc chemistry. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne’s disease. Advantageously, these pure synthetic MAP specific antigens can be produced in non-limiting quantities at low cost. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. Currently available serodiagnostic ELISA tests are based on the use of crude antigens that are not MAP specific and that require a pre-absorption step with antigens of M. phlei to remove cross-reactivity. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.