Structure-based immunopharmacological design of a multi-epitope vaccine candidate against Naegleria fowleri targeting TLR3: a pan-genomic and molecular dynamics approach.

Naegleria fowleri is a highly lethal free-living protozoan responsible for primary amoebic meningoencephalitis (PAM), a rapidly progressive central nervous system infection with a mortality rate exceeding 95%, for which no licensed vaccine or effective preventive therapy currently exists. The absence of prophylactic strategies demands development of novel immunopharmacological interventions. In this study, a comprehensive pan-genomic and structure-guided immunoinformatics approach was employed to design a multi-epitope vaccine candidate against N. fowleri. Four genomes of N. fowleri were subjected to an integrative pan-genomic-immunoinformatics pipeline involving orthologous clustering, subtractive genomics, and immunological relevance screening. From 1427 predicted membrane proteins, 885 antigenic, non-allergenic, and non-toxic candidates were identified. Epitope prediction yielded 7 B-cell epitopes, 26 MHC class I-restricted CTL epitopes, and 22 MHC class II-restricted HTL epitopes, which were assembled into a multi-epitope vaccine construct with immunostimulatory adjuvants. The resulting 320-amino acid construct demonstrated high antigenicity and global population coverage of 91.75%. Structural validation confirmed 98.9% of residues in favorable Ramachandran regions and an ERRAT quality score of 92.484. Molecular docking with Toll-like receptor 3 (TLR3) revealed a highly stable complex with a weighted energy score of - 1475.4 kcal/mol, featuring 24 hydrogen bonds and 5 salt bridges. Molecular dynamics simulations over 100 ns demonstrated structural stability with low RMSD and RMSF values, indicating dynamic stability and coordinated residue motions. Immune simulations predicted strong humoral and cellular immune responses with sustained antibody levels and memory cell generation. Codon optimization achieved a CAI of 0.99 and 53.4% GC content, supporting heterologous expression in Escherichia coli. This multi-epitope vaccine candidate exhibits strong immunogenic potential, structural stability, and favorable interactions with innate immune receptors, positioning it as a promising candidate for experimental validation against N. fowleri infection.