Prediction of potential NS3-based T cell epitopes of hepatitis C virus by in-silico and in-vitro approach.

Due to the absence of an effective vaccine and suboptimal responses to direct-acting antivirals (DAA), Hepatitis C virus (HCV) genotype 3-prevalent in South Asia, the Middle East, and Central Asia-remains a significant public health challenge. This study aimed to identify conserved NS3-based T-cell epitopes of HCV genotype 3 using an integrated in-silico and in-vitro approach. NS3 sequences from 36 HCV-3a infected patients were analysed to generate a consensus sequence, which was translated using the ExPASy-Translate tool and subjected to epitope prediction via the IEDB server. Epitope prediction (percentile rank < 0.5) yielded 119 MHC-I and 436 MHC-II candidates, which were further filtered based on antigenicity (VaxiJen > 0.4), non-allergenicity, non-toxicity, low human homology (E-value > 2), and cytokine-inducing potential (IFN-γ, IL-4, and IL-10). Conservancy analysis across 485 global HCV-3a NS3 sequences shortlisted 11 MHC-I and 6 MHC-II epitopes. Molecular docking demonstrated strong binding affinities with multiple MHC alleles (median RMSD < 2Å). Eleven top-ranked epitopes (6 MHC-I and 5 MHC-II) were synthesised and evaluated using PBMCs from 10 recovered HCV patients. CFDA-SE-based proliferation assays and IFN-γ ELISA confirmed significant T-cell activation (p < 0.05), with higher IFN-γ secretion induced by MHC-I epitopes (1368 ± 151 pg/mL) compared to MHC-II epitopes (1257 ± 64 pg/mL). Primary immune responses were further validated by intracellular IFN-γ staining following stimulation with pooled MHC-I and MHC-II epitopes. Collectively, these findings identify a panel of conserved and immunogenic NS3 epitopes with strong in-vitro validation, supporting their potential utility as core components of genotype-3-specific HCV epitope-based vaccines.