Multi-epitope mRNA Rabies Vaccine Design Using a Reverse Vaccinology Approach Validated by Molecular Dynamics and Binding Energies

Rabies is a zoonotic infection that nearly always results in death in unprotected cases, causing deadly encephalitis. Although inactivated rabies vaccines are available, but they only provide a transient immune response. The need for vaccinations that are safer and more immunogenic has prompted the development of next-generation vaccines. This study utilized a reverse vaccinology approach to create an mRNA vaccine against Rabies by identifying and linking its highly antigenic and immunogenic epitope. Using immunoinformatic tools, potential epitopes for T and B lymphocytes were predicted, with final selection criteria based on the scores of their antigenicity, allergenicity, toxicity and cytokine inducibility. Based on the results obtained, the developed vaccine construct showed antigenicity, remained neutral at physiological pH, exhibited nontoxicity and was nonallergenic, with an estimated maximum coverage of the global population. The mRNA codon optimization ensured efficient expression of the vaccine in host cells. The average GC content of 56.9% and CAI value of 0.93 confirmed the steady expression of the vaccine in the PT7(AG).RV#1.BbsI vector optimized for Homo sapiens (humans) and Mus musculus (domestic mice). Evaluation of various parameters confirmed that our designed mRNA vaccine exhibited a stable structure. A blind docking approach was employed to predict the primary binding mode of the construct with the TLR4 innate immune receptor, resulting in a binding affinity of −279 kcal/mol. Molecular dynamic simulation studies indicated strong interactions with toll-like receptor 4 (TLR4), where the receptor molecule forms a robust binding with the active residues of the epitope during 100 ns simulated intervals. No definitive alterations were seen in either the receptor molecules or the epitopes over this time frame, followed by MMGB/PBSA with high binding affinities validated by Waterswap analysis. We consider these findings to be extremely beneficial for vaccinologists in developing a highly efficient multi-epitope mRNA vaccine for exploratory testing.