*3.1. Strategies to Design Polyepitope T-Cell Antigens*

To stimulate response of CD8+ T-lymphocytes, viral antigens must be presented to CTL precursors not as full-length molecules, but as short peptides (8–12 amino acid residues) in complex with MHC class I molecules. These epitopes are formed from endogenously synthesized viral antigens in the result of proteasome-mediated processing and then are transferred to ER lumina by means of a transporter associated with antigen processing (TAP) proteins where it binds to emerged MHC class I molecules (see for review [31,32]). Since proteasome-mediated processing functions for antigens synthesized intracellularly, a vaccine inducing T-cell response may be designed as DNA vaccine because in this case the CTL vaccine epitopes are presented in the most natural way—through MHC class I-dependent antigen presentation pathway [33].

Unlike stimulation of CTLs, while stimulating CD4+ T-lymphocytes-helpers response, antigen should be presented to those cells in complex with MHC class II molecules. Usually, antigen processing and presentation occurs for extracellular antigens which are delivered in cells via endocytosis and phagocytosis. In this case, antigen processing takes place in the lysosome.

Thus, when designing artificial polyepitope T-cell immunogens capable of inducing responses of CD4+ and CD8+ T-lymphocytes to all epitopes it comprises, it is necessary to provide efficient proteasome- and/or lysosome-mediated processing of the expression product of the target gene by MHC class I and II pathway.

Different strategies can help achieve this goal:


In our study, two artificial polyepitope T-cell immunogens were designed, one of which comprises cytotoxic (CTL) and the other—T-helper (Th) epitopes identified in Ebola virus proteins GP, VP24, VP30, VP35, L, VP40, and NP (Figure 1). Previously we showed that adding ubiquitin to N-terminus of polyepitope antigen induces CD8+ T-cell response more efficiently as compared to adding the signal peptide and the LAMP-1 C-terminal fragment [30]. Therefore, we added N-terminal ubiquitin to the final poly-CTL-epitope construct, and poly-Th-epitope immunogen was designed using N-terminal signal peptide and LAMP-1 C-terminal fragment. N-terminal signal peptide should direct the polyepitope to the endoplasmic reticulum (ER), and C-terminal fragment of LAMP-1 should redirect the polyepitope from the secretory pathway to the lysosome.
