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

L.C. IGLESIAS GARCÍA; J. A. RODRIGUEZ; G. R. BARREDO; J. M. MINOIA; G. ACOSTA; F. ALBERICIO; S. A. CAMPERI

Sitges, Barcelona

Simposio; 36th European and 12th International Peptide Symposium (EPS 2022).; 2022

European peptide society

Envenoming caused by the spider Phoneutria nigriventer constitutes a medical emergency treatable with antivenoms. Its production requires spiders capture and their venom extraction by electrostimulation, a cumbersome, dangerous, and low-yielding method. New approaches are therefore needed for more efficient production to meet the demands. The neurotoxin Tx2-6 (δ-ctenitoxin-Pn2a) is the main responsible for the symptoms of P. nigriventer envenoming in humans. The high stability of this Cys-rich peptide makes it a poor immunogen because its digestion in the antigen-presenting cells, necessary to trigger the 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 Database Analysis 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 acid in their N-terminal. All peptides were analyzed by ESI-MS and RP-HPLC. All the peaks obtained in the mass spectrum corresponded to the synthesized immunogens, 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 synthesized in our laboratory. All the immunogens developed will be assayed to produce P. nigriventer antivenom.