Abstract
Hepatitis C is one of the leading causes of acute and chronic hepatitis worldwide. Immunoinformatics has emerged as a reliable and practical approach for identifying immunogenic regions. We analyzed the HCV genotype 3a polyprotein for antigenicity, allergenicity, and physicochemical characteristics. Four vaccine constructs with possible immunogenic qualities were assembled. B-cell epitopes were examined to predict antigenic determinants, and T-cell epitopes were assessed for MHC class I and II binding. All were linked together using linker peptides, a reliable adjuvant, the PADRE sequence, and a His tag. All models were analyzed for structural refinement, population coverage, molecular docking, molecular dynamics, and immune simulation. The vaccine constructs exhibited antigenicity > 0.6 in E. coli, solubility > 0.8, and GRAVY values between - 0.124 and - 0.518, indicating a hydrophilic nature. The Ramachandran plot showed that 90% of residues were within the allowed regions, confirming structural stability. The vaccine constructs showed more than 99% global coverage and 97% coverage for the Pakistani population. Docking analysis showed strong binding of the Vaccine2-TLR8 and Vaccine4-TLR3 complexes, with docking energies of - 1153.5 kcal/mol and - 1015.8 kcal/mol, respectively. Molecular dynamics simulations revealed a stabilized and consistent complex of TLRs. A strong humoral response was predicted by recording IgG and IgM titers within 5 days, which remained for 35 days. In silico cloning showed successful insertion into the pET-29a ( +) vector. These results indicate that multiple-epitope vaccine constructs have strong binding properties and are good candidates for developing a vaccine against HCV3a.