Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals, including transplant recipients, neonates, and patients with immunosuppression. Despite extensive research, no effective vaccine exists-especially one leveraging mRNA technology. This study aimed to design a safe and immunogenic multi-epitope mRNA vaccine against HCMV using a comprehensive immunoinformatics approach. Glycoprotein B (gB), a highly conserved and immunodominant antigen, was selected as the vaccine target. B-cell, MHC-I, and MHC-II epitopes were predicted based on their antigenicity, immunogenicity, non-allergenicity, and non-toxicity. Of these, three B-cell binding epitopes, 3 MHC-II, and 4 MHC-I binding epitopes of T-cells, along with a toll-like receptor (TLR) agonist and an MHC I- targeting domain (MITD), were rationally assembled to design an mRNA vaccine construct (HCMVV). This construct was evaluated for physicochemical properties, population coverage, structural stability, interactions with innate receptors (TLR 2/4), and simulated immune responses. The vaccine showed high stability and favorable expression profiles, including ten conserved epitopes with broad MHC coverage, especially in South Asian populations. Docking studies indicated a stronger binding affinity for HCMVV-TLR 4 (- 19. 6 kcal/mol) compared to HCMVV-TLR 2 (- 14. 1 kcal/mol), characterized by high affinity and structural complementarity. Normal mode analysis confirmed complex stability, with average deformability per atom index for HCMVV, HCMVV-TLR 2, and HCMVV-TLR 4. Furthermore, molecular dynamics simulation (MDS) over 100 ns using GROMACS confirmed the stability of HCMVV-TLR2 and HCMVV-TLR4 complexes, with RMSD values below 2 Å (HCMVV-TLR4 more stable at <1.3 nm), low RMSF indicating rigidity (except specific flexible regions in TLR4), consistent hydrogen bonds (400-540), compact RoG (<2.7 nm for TLR4), stable SASA (370-430 nmwith slight reduction in TLR4), and PCA revealing dominant conformational motions. Immune simulations predicted strong IgG and T-cell responses, as well as memory formation and efficient antigen clearance. This study introduces a promising mRNA vaccine candidate against HCMV with substantial immunogenic potential, especially for high-risk groups. However, experimental validation, including in vitro expression, in vivo immunogenicity, and protective efficacy studies, is essential to translate these computational findings into clinical applications. 2
