Identification and synthesis of immunogenic peptides to produce Tityus antivenom

RODRÍGUEZ JÉSICA A.; BARREDO VACCHELLI, GABRIELA R.; IGLESIAS-GARCÍA LUCÍA C.; ACOSTA, GERARDO; ALBERICIO, FERNANDO; CAMPERI, SILVIA A.

Simposio; 36th European peptide symposium; 2022

Tityus trivittatus is one of the main scorpions of medical importancein South America. Antivenoms to treat envenoming are producedimmunizing horses repeatedly with scorpion venom which is collectedby a very dangerous, arduous and low-yielding electrostimulation process.Hence, new approaches are needed for more efficient productionto meet the demands. Moreover, peptide neurotoxins,responsable for envenoming symptoms, are low immunogenic Cysrichpeptides. Their high stability hindered their digestion in theantigen-presenting cells, necessary to trigger the adaptive immuneresponse.Beta-mammal Tt1g neurotoxin is the main responsible of T. trivittatusenvenoming symptoms in humans. In this work, the epitopes fromTt1g were identified and immunogenic peptides were designed todevelop synthetic immunogens.The tool ?MHC-II Binding Predictions? from the ?Immune EpitopeDatabase Analysis Resource? was used to identify the epitopes in thetoxin. Its most immunogenic fragment corresponding to its C-terminus,LPNWVKVWERATNRC, was synthesized. Cys was replaced byα-aminobutyric acid to avoid Cys bonds formation. To increase theimmunogenicity, branched peptides were synthesized using Fmoc-Lys(Fmoc)-OH. Also, linear, and branched lipopeptides were developedincorporating palmitic acid in their N-terminal. All peptides were analyzedby ESI-MS and RP-HPLC.All the peaks obtained in the mass spectrum corresponded to the synthesizedimmunogens , demonstrating the high purity of the synthesis.The RP-HPLC analysis of the linear and the palmitoylated epitopesdemonstrated a purity higher than 85%. The branched peptide chromatogramshowed a broad peak, while the branched palmitoylatedpeptide showed two peaks. Probably, the branched palmitoylatedpeptide exists in two forms that convert very slowly between bothconformations. Similar results were obtained with a branched palmitoylatedpeptide of the spider´s Tx2-6 neurotoxin, also synthesized inour laboratory.All the immunogens developed will be assayed to produceT. trivittatus antivenom.