Poster #97 Jennifer Almeyda

Comprehensive computational and evidence-based epitope profiling of Bordetella pertussis

Thalía Silvestre, MSc¹&, Jennifer Z. Almeyda-Tejada, BSc¹&, Sheyla Carmen, BSc¹&, David Tachiquerín, MSc¹, Luis Tataje-Lavanda, PhD²˒³, David Requena, PhD¹* 1 Bioinformatics Group in Multiomics and Immunology. New York, NY, 10016, USA 2 Laboratorios de Investigación y Desarrollo, FARVET. Chincha Alta, Ica, Perú 3 Escuela Profesional de Medicina Humana, Universidad Privada San Juan Bautista. Lima, Perú *Corresponding author: bigmind.us@gmail.com & Equal contribution

Bordetella pertussis has re-emerged as a public health problem, reporting over 43,000 cases in 2024. This might be associated to antigenic variation, yet its immunogenic profile remains poorly understood. In this study, we characterized its immunological landscape by computational assessment and experimental evidence. We annotated 2692 genomes with Prokka and generated a pangenome with Panaroo, identifying 2541 core and 625 soft-core genes, whose proteins were further studied. We benchmarked protein topology prediction tools using UniProtKB annotations. For signal peptides, SignalP-6.0 outperformed Signal-3L (p < 0.0001). For transmembrane regions, TOPCONS2 predictions outperformed DeepTMHMM in length accuracy (p < 0.01) and SOV99 (p < 0.001). We applied the best software and then performed class-I (NetMHCpan, MHCflurry) and class-II (NetMHCIIpan & MixMHC2pred) epitope prediction. We identified the proteins FhaC, FlgE, PtlF, BcrD, BscJ, FimD, FlgI, FlgL and WlbL as epitope-enriched but lacking immunological studies. We developed an epitope prioritization pipeline suitable for bacterial pathogens, which was applied to the total pool of predicted epitopes, resulting in 11 nested HLA-I/HLA-II epitopes for the most frequent South American HLA alleles. Moreover, we tested the performance of our method in correctly identifying experimentally validated epitopes reported in the IEDB database (122 positive/303 negative for HLA-I, 3,452 positive/21,481 negative for HLA-II) significantly increased the likelihood of detecting immunogenic peptides (HLA-I: p < 10⁻⁴, HLA-II: p < 10⁻⁶) than random selection in 10⁷ simulations. These nested epitopes were concatenated in a synthetic construct, with sorting optimized using NeoEpisorter (BigMind Lab). Molecular dynamics confirmed structural stability (RMSD 8-9 Å, Rg 18 Å); and immune simulations predicted CD4+ T-cell response. In summary, this study identified conserved proteins of Bordetella pertussis with immunological relevance, expanding the potential antigenic landscape and providing insights into its pathogenicity, immunodiagnostics, and vaccines development.