*3.3. Design of Poly-Th-Epitope Ebola Virus*

To achieve the most efficient induction of T-cell immune response, one should induce not only responses of CD8+ but also CD4+ T-lymphocytes; therefore, in the following steps, we constructed poly-Th-epitope fragment (EV.Th). We used Th-epitopes predicted for humans and showing the broadest specificity regarding HLA class II molecules. For the purpose, TEpredict [25] predicted Th-epitopes in Ebola virus proteins. PolyCTLDesigner [26] was used to select eight fragments of the length of 35–40 amino acid residues comprising the most of the Th-epitopes with the broadest specificity regarding different HLA class II allomorphs. N-terminus of the selected peptides was extended up to 5 amino acid residues as compared to the beginning of the first epitope, and C-terminus—up to 5 amino acid residues as compared to the end of the last epitope (Table 2).

Additionally included at C-terminus of the construct: universal Th-epitope PADRE (PAn DR Epitope)—AKFVAAWTLKAAA; marker epitope EPFRDYVDRFYKTLR of p24 HIV-1 protein recognized by monoclonal antibodies 29F2, and a C-terminal fragment of LAMP-1 protein— RKRSHAGYQTI. According to the literature, adding the signal peptide concurrently with LAMP-1 C-terminus fragment to the target antigen raises the level of CD4+ T-lymphocyte response significantly [50–53]. As a signal peptide, we selected the sequence of an N-terminal fragment of Ebola virus surface glycoprotein comprising MGVTGILQLPRDR leader peptide. Using the SignalP server [54] we predicted that the leader peptide in the designed artificial polypeptide is functional and should efficiently split out. Poly-Th-epitope antigen EV.Th was designed using K/R-K/R spacer sequences that form cleavage sites by lysosomal cathepsins [39,40]:

*MGVTGILQLPRDR*—FKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKL—**RR**—TNTNHFN MRTQRVKEQLSLKMLSLIRSNILKFINKLDA—**RR**—LTLDNFLYYLTTQIHNLPHRSLRILKPTFK
