Samina Baig ¹ Assel Berikkara ² Ramsha Khalid ¹ Syed A. Subhan ¹ Tanveer Abbas ¹ Syed Hani Abidi ^2*

• ¹ University of Karachi, Karachi, Sindh, Pakistan
• ² Nazarbayev University, Nur-Sultan, Kazakhstan

Background: HCV genotypes are 30–35% polymorphic at the nucleotide level, while subtypes within the same genotype differ by nearly 20%. Although previous studies have shown the immune escape potential of several mutations within the HCV proteins, little is known about the effect of genotype/subtype-specific gene polymorphism on T-cell immunity. Therefore, this study employed several in silico methods to examine the impact of genotype/subtype-specific polymorphisms in Core, NS3, NS5A, and NS5B sequences on T cell epitope processing and HLA-epitope interactions. Methods: For this study, 8,942, 17,700, 14,645, and 3,277 HCV Core, NS3, NS5A, and NS5B sequences, respectively, from eight genotypes and 21 subtypes were retrieved from the Los Alamos HCV Database. Next, the NetCTL tool was employed to predict Cytotoxic T Lymphocyte (CTL) epitopes based on combined proteasomal cleavage, TAP efficacy, and HLA class I receptor binding scores. PEP-FOLD was used to model selected epitopes, followed by peptide-HLA docking using HPEPDOCK. Finally, molecular dynamics simulations were conducted for 200 ns using Desmond software to analyze differences in HLA-epitope (from different HCV genotypes) interaction kinetics and dynamics. Results: A total of 3,410, 8,054, 6,532, and 14,015 CTL epitopes were observed in the HCV Core, NS3, NS5A, and NS5B sequences, respectively. Significant genotype/subtype-specific variations in CTL values and docking scores were observed among NS3, NS5A, and NS5B proteins. In silico results reveal that epitopes from genotype 6b (NS3), 6d/r (NS5B), 6o and 6k (NS5A) exhibit higher immunogenicity than other genotypes, forming more energetically stable complexes with host receptors. These epitopes, compared to those from the same positions but different genotypes, showed binding energies of -144.24 kcal/mol, -85.30 kcal/mol, and -43 kcal/mol, respectively. Over a 200 ns MD simulation, GT 6b and 6d/r epitopes displayed up to a 40% stronger binding energy with the HLA receptor. These findings suggest that patients infected with GT 6 may experience enhanced T cell responsiveness and broader immunogenicity. Conclusion: Genotype/subtype-specific polymorphism in HCV may result in altered immune responses by modulating T-cell epitope processing and interaction with HLA receptors. Further experimental studies can be performed to confirm the effect of genotype/subtype-specific polymorphisms on T cell-mediated immune response.