*3.4. Immune Response to Administration of Modified MSCs to Mice Exceeds Immune Response to Plasmid*

Our next goal was to evaluate the immune response in mice immunized with the mMSC. Comparative analysis of the humoral immune response in mice showed that mMSC (group 1) induced the formation of antibodies to all viral proteins encoded by the plasmid. Levels of antibodies to NS3, NS4, and NS5A were on average 40-fold higher than in group 3 (immunized with the pNS3-NS5B

plasmid) (Table 1). In contrast, levels of antibodies to NS5B were higher in mice immunized with the plasmid than with mMSC (Table 1). The distribution of antibody isotypes for HCV proteins differed between the groups: in groups 1 and 3, the antibodies belonged mainly to the IgG2a isotype; in group 1, IgG1 antibodies to the NS3 protein were also detected. After introduction of naïve MSCs (group 2), IgG2a antibodies were not found, while IgG1 antibodies were detected in only four out of ten mice and were present at a low level. Therefore, differences with the control were not statistically significant.

**Table 1.** The levels of anti-HCV antibodies in the sera of mice receiving two injections of mMSC, MSC, or plasmid.


Four pools of recombinant HCV proteins (described in Materials and Methods) were used as sorbents in ELISA to evaluate antibody production; IgG1 and IgG2a are antibody isotypes. Values show the geometric mean titer ± SD of three measurements done from three independent experiments. \* *p* < 0.05 compared to control; **#** *p* < 0.05 compared to all groups. Numbers of animals in each group that developed antibodies to the HCV proteins to the total numbers of animals are given in brackets.

To assess the cellular response of lymphocytes in vitro, we used recombinant proteins from the HCV non-structural region as specific stimulants. For a negative control, we also assessed the response to the core protein that was not encoded by the pNS3-NS5B plasmid and in mMSCs. For a positive control, ConA was used for stimulation.

All tested non-structural HCV proteins stimulated proliferation of splenocytes in groups 1 and 3, SIs statistically significantly (*p* < 0.05) differed from SIs in groups 2 and 4 (Figure 4a). In group 1, SIs exceeded those in group 3 by 2.5–6.1 times, on average by 4.2 ± 1.6 times. The greatest proliferative response was caused by NS5B, when SI reached 27.

Stimulated lymphocytes secreted IFN-γ (Figure 4b). In group 1, the response was obtained to all specific antigens; the highest cytokine concentration (over 1.7 ng/mL) was stimulated by NS5B. In group 3, the production of IFN-γ was induced by all proteins except NS5A; the NS4 protein showed the highest activity. In groups 1 and 3, the cytokine levels secreted in response to specific antigens varied in a wide range from 3.5 to 60-fold, being on average 30-fold higher than in the case of groups 2 and 4.

In ELISpot assay, the average number of IFN-γ synthesizing cells in response to four specific stimulants in groups 1 and 3 was significantly higher than that in the control groups (Figure 4c). Differences in signal intensity between groups 1 and 3 were 2.6 ± 0.2. The NS5B protein exhibited higher activity than other virus antigens. It should be noted that group 2, which was administered to naïve MSCs, demonstrated immune responses to HCV proteins in contrast to group 4, but their intensity was much lower than in the case of the transfected cells in group 1 (Figure 4b,c).

The ELISpot reaction was also performed with splenocytes from mice that were administered mMSC treated with IFN-γ. A number of IFN-γ producing cells in this group was 11-fold lower than those in group 1. It is noteworthy that the number of spots for NS3 was 13.3 ± 7.1%, for NS4—7.1 ± 2.6%, for NS5A—7.6 ± 1.5%, for NS5B—8.9 ± 4.2% of the corresponding values in group 1.

**Figure 4.** A comparative analysis of the cellular immune response in mice to HCV proteins in vitro after immunization with modified MSC, naïve MSC, and plasmid. Four groups (Gr) of mice were injected twice with mMSC (Gr1), non-transfected MSC (Gr2), plasmid pcNS3-NS5B (Gr3), or saline (Gr4). To assess the cellular response of lymphocytes in vitro, we used purified recombinant proteins from the non-structural region of HCV, which were combined into four pools (NS3, NS4, NS5A, and NS5B). A recombinant HCV core and medium alone were used as negative controls (negative). (**a**) Results of T-cell proliferation are expressed as stimulation indexes (SI); the IFN-γ production by splenocytes in response to HCV antigens was assayed as cytokine concentrations in culture fluids by ELISA, expressed as pg/ml (**b**), or as the number of IFN-γ synthesizing cells by ELISpot in the number of spot forming cells (SFC) per 10<sup>6</sup> cells (**c**). Values on each diagram are means <sup>±</sup> SD of three measurements done in three independent experiments. \* *p* < 0.05 compared to control; # *p* < 0.05 compared to all groups.

Assessment of the results of T-cell proliferation and ELISpot assays showed that mouse groups 1 and 2, which received MSCs, had a higher level of spontaneous cellular response as well as the response to ConA, compared to groups 3 and 4 (*p* < 0.05, Table 2). Differences in IFN-γ production during ConA stimulation between groups were not statistically significant.

**Table 2.** Spontaneous and concanavalin A (ConA)-induced cellular response of lymphocytes from immunized mice in vitro.


Values are means ± SD of three measurements done in three independent experiments. \* *p* < 0.05 compared to Gr3 and Gr4.
