Falcone, F.H.

Hossain, M.S.

Thomas, Lian F.

Toye, Philip G.

CGSpace

Development of a fluorescent RBL reporter system for diagnosis of porcine cysticercosis          Md. Shahadat Hossain1,2, Philip Toye2, Lian Thomas2,3, Franco H. Falcone1                                1Justus Liebig University Giessen, 2 International Livestock Research Institute (ILRI), 3 University of  Liverpool Created with BioRender Poster BuilderBackground & Objectives→Porcine cysticercosis is caused by a zoonotic neglected tropical disease parasite, Taenia solium in pig.→PCC reduces pork value, affects food security and livelihood of  pig farmers.→Tongue palpation and meat inspection are most widely used diagnostic methods.→Serological diagnosis is based on IgG, characterized by low sensitivity.→IgE plays the central role in metazoan parasitic infections.⊕ Objective 1: Development and characterization of  porcinized IgE reporter cell lines which can bind pig IgE.⊕ Objective 2: Selection, cloning, and recombinant expression of  candidate  allergens of  T. solium, followed by their validation as diagnostic antigens.Test Principle⊕ Porcinized  IgE reporter system created using Rat basophil leukaemia (RBL) cells stably transfected with neuropeptide Y monomeric red fluorescent protein fusion (RBL NPY-mRFP), located in granules (Fig.1).α ααRat IgEβ γPig IgE Pig IgEγβ γ γβ γγChimeric Pig/Rat FcεRIα Pig FcεRIαRat FcεRIαFig. 1 Creation of  chimeric pig/rat FcεRIα cell line⊕ Porcinized reporter system incubated overnight with pig-IgE  followed by stimulation with allergens, results in IgE crosslinking, by allergens, followed by degranulation of  reporter cells (Fig.2). βγγαβγ γα βγγαβγ γαAllergensIgEFcεR1IgERBL NPYmRFP-pig/rat FCεRIα reporter systemMeasures red florescent protein (mRFP), released by stimulated cells in supernatant after 45 minutes of incubation  (Ex/Em 530/590 nm)Fig.2 Activation of  the porcinized IgE reporter system by IgE-allergen interaction. Crosslinking of  receptor-bound IgE by allergen induces degranulation and mRFP release into supernatant.MethodsSelection of  putative IgE-binding antigens expressed by tissue migrating oncosphere stage for cloning followed by sequence verification of  successful clones; allergenicity prediction of  diagnostic allergens.Published proteomic and transcriptomic dataA A TG C GT CT GSequence analysis Protein motifs (e.g. EF-Hand, potease signatures)Allergen Databases (e.g. AllFam, Allergome, AlgPred 2.0)Tyagi et al. predicted helminth 'allergens'Nucleofection of  NPY-mRFP reporter with pig high affinity IgE receptor (alpha chain)Antibiotic selection of  stable transfectantClonal selection of  best porcine IgE reportersSuitability as diagnostic antigens (Specificity, Sensitivity, ROC curves)T A ATC CA T TAG GTACGCGATCGTA pTTAllergensCodon optimizationCloningHEK-293 6E cell culture Transfection Shaking  IncubatorRecombinant expression of  allergens in HEK293-6E cells followed by chromatographic purificationResults⊕ Five candidate diagnostic T. solium oncospheral allergens identified through bioinformatics analysis (Fig.3) and allergenicity confirmed by AlgPred 2.0. Fig. 3 Candidate allergens of  T. solium ⊕ Three HEK293-6E cell supernatants transfected T. solium allergens  showed expected protein band size in Western blot analysis (Fig.4).⊕ K0A0S9: 19.8 kDa , Q2XNL7: 20 kDa,and W8P1J2: 22.3 kDa.10172634435572kDa 1 2 3 4 5 6 719.8 kDa 20 kDa22.3 kDa Fig. 4 Successful protein expression of  transfected allergens in Western blot. Lane 1: protein ladder; Lane 2: negative control, Lane 3-7: transfected T. solium allergens .Conclusion✓ Stably transfected chimeric pig/rat FcεRIα reporter system has been created(Fig.5)✓ Successful cloning and sequence verification of  four recombinant T. solium allergens in expression vector✓ Successful recombinant expression Fig. 5 Stable transfection and PCR confirmation of  chimeric pig/rat FcεRIα cell line. Fluorescence microscopy showing red fluorescence from transfected cells. In PCR, RBL-2H3 cells used for negative control (SS-nc). SS: chimeric pig/rat FcεRIα, MW is a 100 bp ladder.Outlook● Further expression and purification of  candidate diagnostic allergens.● Validation and assessment of  Porcinized RBL reporter system with diagnostic allergens.● Screening of  infected and non-infected pig sera with reporter system.● Determination of  specificity and sensitivity.Commissioned by the German Federal Ministry for Economic Cooperation and Development (BMZ) and carried out by ATSAF e.V. on behalf of the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH. 

Development of a fluorescent RBL reporter system for diagnosis of porcine cysticercosis

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Development of a fluorescent RBL reporter system for diagnosis of porcine cysticercosis

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