Identification and synthesis of epitopes from a Phoneutria nigriventer toxin to produce immunogens

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

Simposio; 36th European peptide symposium; 2022

Envenoming caused by the spider Phoneutria nigriventer constitutes amedical emergency treatable with antivenoms. Its production requiresspiders capture and their venom extraction by electrostimulation, acumbersome, dangerous, and low-yielding method. New approachesare therefore needed for more efficient production to meet thedemands. The neurotoxin Tx2-6 (δ-ctenitoxin-Pn2a) is the mainresponsible for the symptoms of P. nigriventer envenoming in humans.The high stability of this Cys-rich peptide makes it a poor immunogenbecause its digestion in the antigen-presenting cells, necessary to triggerthe adaptive immune response is hindered.In this work, the epitopes of the neurotoxin Tx2-6 were identified, and immunogens for Phoneutria bites antivenom production were designed and Synthesized.The "MHC-II Binding Predictions" tool of the "Immune Epitope DatabaseAnalysis Resource" program was used to identify the epitopes.The most immunogenic zone corresponding to the C-terminal, GYFWIAWYKLANCKK,was synthesized in solid phase using Fmoc/tBu chemistry. The Cys was replaced by α-aminobutyric acid to avoid disulfide bonds formation. To increase the immunogenicity, branched peptides were synthesized using Fmoc-Lys (Fmoc)-OH. Also, linear, and branched lipopeptides were developed incorporating palmitic acidin their N-terminal. All peptides were analyzed by ESI-MS and RPHPLC.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 epitope and the palmitoylated epitope demonstrated a purity higher than 85%. The branched peptide chromatogram showed a broad peak, while the branched palmitoylated peptide showed two peaks. Probably, the branched palmitoylated peptide exists in two forms that convert very slowly between both conformations. Similar results were obtained with a branched palmitoylated peptide of the scorpion´s Tt1g neurotoxin, also synthesizedin our laboratory.All the immunogens developed will be assayed to produce P. nigriventer antivenom.