NS3 epitope-decorated nanoparticles produced in bacteria trigger potent T cell immunity against hepatitis C virus

The highly conserved human leukocyte antigen-A2 (HLA-A2)-restricted epitope NS3-1073 represents a promising candidate for a therapeutic vaccine against hepatitis C virus (HCV). In this study, we engineered a set of fusion proteins based on the artificial self-assembling peptide (SAP), which were expressed in <i>Escherichia coli</i> and spontaneously self-assembled into nanosized particles displaying HCV epitopes, including NS3-1073. To enhance immunogenicity, we incorporated the T helper epitope PADRE into the construct. Alpha-helical linkers were introduced between SAP and the epitopes to facilitate proper protein folding. Notably, a helical linker with a high supercoiling propensity enabled soluble expression of the fusion protein containing both the NS3-1073 and PADRE epitopes, allowing purification of the <i>in vivo</i>-formed nanoparticles by metal affinity chromatography. Human dendritic cells derived from peripheral blood monocytes showed robust activation in response to the fusion proteins and preferentially stimulated T lymphocytes toward a Th1-biased immune response. In mice, immunization with nanoparticles carrying NS3-1073 induced splenocyte proliferation in response to <i>in vitro</i> stimulation with a mixture of NS3 peptides. These results demonstrate that recombinant nanoparticle-based carriers presenting the NS3-1073 epitope can be produced in bacterial systems and hold strong potential as a foundation for a therapeutic HCV vaccine.