Développement d'un test basé sur les aptamères pour le séro-diagnostic des trichinelloses

Objectives The aim is to develop an innovative and highly specific approach to detect Trichinella infected animals (or humans) based on aptamers directed against Trichinella whole larvae and/or against larval molecules (proteins, carbohydrates, ...). It will allow an early detection of infected animals or patients using blood samples. This in turn will result in: - implementation of serological monitoring programmes to reduce the costs of the current Trichinella testing schemes, and at the same time guarantee removal of infected animals from the food chain ; - early onset therapy in humans avoiding the parasite installation in muscles. Methods An initial random DNA library already available at the French NRL will be used. In order to select specific targets, two selection strategies based on the method of "Systematic Evolution of Ligands by Enrichment" (SELEX) will be adapted to Trichinella. The first is a method of Whole Larvae-SELEX, very innovative since the technique has never been performed on whole larvae; the second is a Protein-SELEX method, already used for the selection of aptamers against protozoa. Two subsequent positive selection steps based on the first strategy (whole larvae-SELEX) will be performed in order to greatly reduce the number of aptamers in the DNA library. Then ten more steps of positive selections will be implemented by decreasing the aptamer quantity and the contact time between the previous aptamers with the larvae to increase specificity. Some aptamers may be able to bind to Trichinella but could also cross-react with other parasites. At each of the 10 positive selection steps, negative selections will be made on other nematodes larvae. Only the aptamers that do not link to these other nematodes will be preserved. Obtaining other species will be easy since the French partner is 1) located on the campus of the National Veterinary School of Alfort and benefits from numerous samples provided by veterinary colleagues (Ascaris suum, Toxocara canis, T. cati, ...); and 2) co-leader of the French Nematode Network (REFNEM). The aptamers selected on whole larvae will be used for the development of serological tests. Nevertheless, there is still a risk of cross-reactions during co-infection with other parasites. These cross-reactions will be tested on patient and pig sera known to be infected with other parasite species but not with Trichinella. If it turns out that false positives are obtained, a Protein-SELEX strategy will be performed. Recombinant proteins specific for Trichinella and expressed early in the infection, such as NBL1, L20HTs1, L20HTs3, but also later proteins such as the p49, 3,6-dideoxy-D-arabino-hexose or beta-tyvelose, a specific carbohydrate of Trichinella, will be targeted by aptamer selections either from the first selected aptamers on whole larvae or from a new library. At least 8 steps of positive selections will be made by decreasing the amount of recombinant proteins and the aptamer-protein contact time to gain specificity. For each strategy, the different aptamers present in the last selection (different nucleotide sequences) may be quite numerous. They will then be identified by cloning/sequencing and / or by NGS. Then, small DNAs corresponding to these sequences will be synthesized in order to carry out the first Aptamer-Based ELISA tests on patient and pig sera that have been collected during infections versus healthy sera. Also, both the French and German NRLs have defined Trichinella positive and negative pig sera available. Different combinations of aptamers will also be tested to gain sensitivity. The selected aptamers and a protocol will be sent to the German (veterinary samples) and Canadian partners (human samples) that will test their available sera in a blinded approach. Test parameters such as sensitivity, specificity, reproducibility and robustness will be determined.

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