Design and Expression of Multiepitope Constructs Using mRNA Vaccine Technology.

is a parasitic trematode responsible for fascioliasis, a significant zoonotic disease affecting livestock worldwide, as well as humans. This study identifies peptides with potential for use in vaccines against and validates multi-epitope constructs from those peptides in vitro. Putative protein sequences derived from the genome of were integrated with phase-specific transcriptomic data to prioritize highly expressed proteins. Among these, extracellular proteins were selected using DeepLoc 2.0 and strong binding affinities across diverse human and murine alleles were predicted with the IEDB MHC II tool. Peptides were further selected based on their toxicity, immunogenicity, and allergenicity. Finally, 55 high-priority candidates were obtained. To express these candidates, mRNA constructs encoding various combinations of these peptides were designed, synthesized using in vitro transcription with T7 or SP6 RNA polymerases, and transfected into cells for expression analysis. SP6 polymerase produced proper capping using CleanCapAG and was far superior in transcribing peptide constructs. Peptides fused in frame with eGFP were expressed efficiently, particularly when peptides were positioned at the 3' terminus, opening a new field of peptide vaccines created using mRNA technology.