*3.6. Immunogenicity Study of DNA-Vaccine Constructs Encoding Multiple T-Cell Epitopes of Ebola Virus*

Immunogenicity of the target DNA vaccine constructs was evaluated regarding their capacity to induce a T-cell response in BALB/c mice 14 days after the third immunization. The level of T-cell immune response was detected using IFNγ-ELISpot and ICS.

ELISpot results (Figure 4) demonstrate that the induction of specific response was registered in both experimental groups [pE-CTL+pE-Th] and [pE-CTL], especially in the animal group immunized with a mix of target DNA vaccine constructs [pE-CTL+pE-Th]. Significant differences from both negative controls were observed only in [pE-CTL+pE-Th] group (Table 3).

The capacity of vaccine constructs to induce IFNγ-producing CD4+ and CD8+ T-cells was tested by ICS after stimulating splenocytes with specific peptides. The results of ICS (Figure 5) revealed that statistically significant difference from control (Table 4) was demonstrated by IFNγ-producing CD8+ T-lymphocytes in animal groups immunized both with pEV.CTL and a mix (pEV.CTL + pEV.Th) DNA vaccines as well as by IFNγ-producing CD4+ T-helpers in the group immunized with only a

mix of vaccine constructs (pEV.CTL + pEV.Th). The maximal responses of IFNγ-producing CD8+ T-lymphocytes (*p* = 0.024) and CD4 + T-cells (*p* = 0.012) were registered in the animal group immunized with a mix of vaccine constructs. This is believed to be caused by the synergistic effect of CD8+ and CD4+ T-lymphocytes.

**Figure 4.** The results of IFNγ-producing T-cell count in IFNγ-ELISpot assay in BALB/c mice immunized with DNA-vaccine constructs encoding target immunogens (n = 6 for phosphate buffer solution (PBS) control group and n = 10 for the other groups). The figure represents spot count (i.e., IFNγ-producing T-cells) in different experimental and control animal groups.

**Table 3.** Results of statistical data analysis in ELISpot.

**Figure 5.** The results of IFNγ-producing CD4+ (A) and CD8+ (B) T-cell count using intracellular cytokine staining (ICS) approach in BALB/c mice immunized with DNA vaccine constructs encoding target immunogens (n = 5).

To design the target antigens, we used PolyCTLDesigner software that we had developed for rational design of artificial polyepitope vaccine constructs [26]. It enables us to calculate amino acid sequence of polyepitope antigen by detecting the best spacer sequences for each pair of epitopes and optimal relative positions of epitopes in the construct considering state-of-the-art knowledge about the specificity of proteasomal processing of antigens and interaction between peptides and TAP. The findings revealed that the designed artificial DNA vaccine constructs encoding CTL and Th-epitopes of Ebola virus antigens provide expression of the target genes, as well as induce virus-specific responses of CD4+ and CD8+ T-lymphocytes in immunized mice.


**Table 4.** Statistical analysis results obtained using intracellular cytokine staining (ICS).
