Activation of Human Dendritic Cells by Nanoparticles Carrying CTL Epitopes of Non-Structural Proteins of Hepatitis C Virus

Author(s): Kuprianov Victor, Lyudmila Nikolaeva, Anna Zykova, Anna Dedova, Artemiy Vakhrameev, Nikolai Ravin

The aim of this study was to produce immunogenic nanoparticles carrying cytotoxic T lymphocyte (CTL) epitopes of hepatitis C virus non-structural proteins. We have obtained recombinant proteins forming virus-like particles and containing the sequences of self-assembling peptides (SAP), PADRE, and CTL epitopes. Using atomic force microscopy (AFM), the size of thus obtained nanoparticles was shown to be dependent on number of CTL epitopes located at the C terminus of recombinant proteins. Recombinant protein aggregating into virus-like particles (VLP) consisted of SAP and a helical linker. It formed 16-18 nm homogeneous particles, as shown by AFM. VPL-PE1, which differs from VPL by the presence of PADRE and CTL epitopes from NS3, forms 25-30 nm nanoparticles. The VLP-PE2 protein, different from VLP-PE1 by the presence of NS4a and NS4b CTL epitopes, formed 70-80 nm nanoparticles. The size of nanoparticles depended on the presence of SAP and the number of inserted epitopes. These nanoparticles activated human dendritic cells (DCs) that, in turn, stimulated autologous T lymphocytes. The proliferative activity and interferon-γ (IFN-γ) production by the stimulated T lymphocytes were evaluated by their secondary stimulation with commercially available mixture of peptides from non-structural proteins of hepatitis C virus. The greatest stimulating effect on T lymphocytes was exerted by DCs activated by nanoparticles, consisting of the recombinant mosaic protein with SAP at the N terminus and CTL epitopes of NS3, NS4a, and NS4b proteins at the C terminus. The presence of SAP in recombinant proteins’ sequences increased their immunogenicity.