Semester of Graduation
Spring 2025
Degree
Master of Science (MS)
Department
School of Animal Sciences
Document Type
Thesis
Abstract
Equine Herpesvirus-1 (EHV 1) is a worldwide significant pathogen that causes respiratory illness, abortion, and neurological disorders in equine. Current vaccines, including live attenuated, inactivated, and subunit platforms, do not prevent viral latency, mucosal shedding, or cross-strain immunity, requiring alternative approaches. To address these gaps, this project explores the design, synthesis, and in vitro testing of an mRNA vaccine targeting immunogenic EHV 1 glycoprotein (gB, gC, gD, gG, and gM). Epitopes were computationally predicted using the Immune Epitope Database (IEDB), codon-optimized for Bos taurus, and cloned into pUCIDT vectors using SP6/T7 promoters. In vitro transcription (IVT) used nucleotide modifications (pseudouridine, 5-methylcytidine) and capping methods (Cap 0/Cap 1) to improve stability and translation efficiency.
The SP6 and modified T7 promoters effectively produced RNA transcripts, with modified T7 yielding 2.3 times more than SP6 (p = 0.002). However, Western blot analysis of lysate and media from transfected HEK293 cells indicated no detectable protein expression, indicating translation efficiency issues, RNA secondary structure interference, or rapid protein breakdown. Modified nucleotides (such as pseudouridine) increased RNA stability but failed to eliminate translation challenges. Statistical analysis revealed no significant variation in RNA concentrations between Cap 0 and Cap 1 (p = 0.1272), but nucleotide modifications significantly affected RNA synthesis (p = 0.0068).
This study shows the potential of mRNA technology for EHV 1 vaccines while highlighting limitations in protein expression. Future studies should focus on RNA secondary structure optimization, exploring lipid nanoparticle delivery techniques, and validating designs in equine cell lines or in vivo models. These findings advance the study of mRNA vaccines for veterinary use by offering a foundation for quick, flexible solutions for evolving equine diseases.
Date
3-24-2025
Recommended Citation
Devkota, Bhawana, "The Design, Construction, And Testing of mRNA Vaccine Against Equine Herpes Virus" (2025). LSU Master's Theses. 6106.
https://repository.lsu.edu/gradschool_theses/6106
Committee Chair
Cooper, Richard K.
Included in
Animal Diseases Commons, Animal Sciences Commons, Biology Commons, Biotechnology Commons, Immunology and Infectious Disease Commons, Molecular Genetics Commons, Pharmaceutics and Drug Design Commons, Veterinary Medicine Commons, Virology Commons, Virus Diseases Commons
