Quasispecies-guided E1 and E2 epitopes for rational design of a multi-epitope HCV vaccine

Background Hepatitis C virus (HCV) remains a major global health burden, infecting approximately 58 million individuals worldwide. Despite the high efficacy of direct-acting antivirals, the absence of a preventive vaccine and the extensive genetic diversity of HCV—manifested through seven genotypes and intra-host quasispecies populations—continue to impede eradication efforts. The envelope glycoproteins E1 and E2 are primary targets for vaccine development; however, their hypervariable regions pose a significant challenge to the design of broadly protective immunogens. Methods A quasispecies-guided immunoinformatics pipeline was developed to rationally design a chimeric multi-epitope vaccine against HCV. A total of 41 E1 and 33 E2 sequences retrieved from UniProtKB were subjected to multiple sequence alignment using Clustal Omega and variability analysis via BioEdit. Hypervariable segments were reconstructed into a virtual variant library incorporating dominant amino acid substitution frequencies. Epitope prediction for linear B-cell lymphocyte (LBL), cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) candidates was performed using IEDB-based tools (Emini, NetMHCpan 4.1, and NetMHCIIpan 4.1). All epitopes were screened for antigenicity (VaxiJen v2.0), immunogenicity, allergenicity (AllerTOP v2.0), and toxicity (ToxinPred2). The final construct incorporated selected epitopes linked by AAY, GPGPG, and KK linkers, with the cholera toxin B subunit (CTB) as an N-terminal adjuvant via an EAAAK linker. Structural modeling was performed with AlphaFold2, refined via GalaxyRefine, and validated using ERRAT, PROCHECK, and ProSA-web. Molecular docking against TLR4 was conducted with ClusPro 2.0, and a 50 ns molecular dynamics (MD) simulation was carried out using GROMACS. In silico immune response was evaluated using the C-ImmSim server. Codon optimization was performed for expression in Escherichia coli. Results Five LBL, four CTL, and two HTL epitopes passed all immunological and safety filters. The combined epitope set achieved a global HLA population coverage of 99.26%. The 239-amino-acid chimeric construct demonstrated an antigenicity score of 0.6956 (VaxiJen) and 0.9215 (ANTIGENpro), was classified as non-allergenic and non-toxic, and exhibited a predicted solubility of 0.509. Structural validation confirmed 97.6% of residues in Ramachandran-favored regions and an ERRAT quality factor of 89.03%. Molecular docking revealed a binding free energy of − 19.4 kcal/mol and a dissociation constant of 5.6 × 10⁻¹⁵ M with TLR4. MD simulation confirmed complex stability over 50 ns. In silico immunization predicted robust Th1/Th2 cytokine responses, elevated IgG titers, and sustained immunological memory. Conclusion This study presents the first quasispecies-informed multi-epitope HCV vaccine construct, demonstrating that integrating variant-level sequence diversity into the epitope selection pipeline yields immunogens with enhanced mutation resilience and broad cross-genotypic potential. The construct's favorable structural, thermodynamic, and immunogenic properties warrant its advancement to experimental validation.