How Does Inter-Epitope Spacer Variation Within Artificial Immunogens Based on T-Cell Epitopes of Tick-Borne Encephalitis Virus Affect Immunogenicity?

Background/Objectives: In recent years, researchers have directed considerable attention toward the activation of the T-cell response in the development of vaccines against viral infections, including the tick-borne encephalitis virus (TBEV). A particularly promising approach to developing effective and safe T-cell vaccines involves the use of artificial multi-epitope immunogens. The selection of spacers that link epitopes within a construct can have a significant effect on the immunogenicity of multi-epitope constructs. The objective of this study was to design multi-epitope TBEV immunogens using various spacers and to evaluate their immunogenicity. Methods: The present study involved the design of three multi-epitope immunogens, which were developed based on T-cell epitopes from TBEV proteins. The AG1-ub construct contained optimized alanine spacers, the AG2-ub construct contained GPGPG spacers, and the AG4-ub construct contained no spacers. Three DNA vaccines encoding the designed immunogens were subsequently produced. To assess the immunogenicity of the constructs, BALB/c mice were immunized with the designed DNA vaccines via electroporation. Results: The ELISpot assay demonstrated that DNA vaccines encoding AG1-ub and AG4-ub induced a significant number of IFN-γ-producing cells. The DNA vaccine encoding AG2-ub, a multi-epitope with GPGPG spacers, exhibited low immunogenicity, potentially attributable to inadequate processing or misfolding of the AG2-ub protein, thereby affecting the molecule's structure. Conclusions: The data provided in this study on the effect of spacers on the immunogenicity of multi-epitope constructs can be used to optimize the design of vaccine candidates. However, further research is needed to assess their clinical potential.