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. 2023 May 30;12(6):792.
doi: 10.3390/biology12060792.

Human Mesenchymal Stem Cells Modified with the NS5A Gene of Hepatitis C Virus Induce a Cellular Immune Response Exceeding the Response to DNA Immunization with This Gene

Olga V Masalova  1 , Ekaterina I Lesnova  1 , Vladimir A Kalsin  2 , Regina R Klimova  1 , Natalya E Fedorova  1 , Vyacheslav V Kozlov  1 , Natalya A Demidova  1 , Kirill I Yurlov  1 , Mikhail A Konoplyannikov  2   3 , Tatyana N Nikolaeva  1 , Alexander V Pronin  1 , Vladimir P Baklaushev  2 , Alla A Kushch  1
Affiliations

Human Mesenchymal Stem Cells Modified with the NS5A Gene of Hepatitis C Virus Induce a Cellular Immune Response Exceeding the Response to DNA Immunization with This Gene

Olga V Masalova et al. Biology (Basel). .

Abstract

Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.

Keywords: DNA immunization; HCV vaccine; cellular immune response; genetically modified MSCs; hepatitis C virus (HCV); human mesenchymal stem cells (MSCs); memory T cells; myeloid derived suppressor cells (MDSCs); non-structural HCV NS5A protein.

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Conflict of interest statement

The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Cell doubling times for MSCs. Cell doubling times for MSCs from various sources were calculated based on the cell counts at the fourth passage. Triplicates of each group were investigated in every donor sample. The results represent the mean ± SD; * p < 0.05 indicates the statistical significance of DP-MSCs vs. other MSCs (One-way ANOVA and Tukey’s multiple comparison tests).
Figure 2
Figure 2
Dental pulp-derived MSCs characterization. FACS analysis of the DP-MSCs. Blue histograms represent the isotype-specific Ig control; red histograms represent FITC/PE-conjugated appropriate antibodies (a). MSCs’ differentiation potential analyzed by their staining with Oil Red O (adipogenic lineage, A), Alizarin Red (osteogenic lineage, B) and Alcian Blue (chondrogenic lineage, C) (b). Magnification ×200.
Figure 3
Figure 3
Transfection efficiency of Huh7.5 human hepatoma cells (A,D) and human MSCs from the adipose tissue (B,E) and dental pulp (C,F). GFP fluorescence 48 h after the cell transfection with the chimeric pcNS5A-GFP plasmid; scale bars, 100 µM (A–C), and 25 µM (D,E).
Figure 4
Figure 4
Differences in the level of cytokine secretion by human mesenchymal stem cells before and after the transfection. Concentrations of cytokines secreted by MSCs obtained from different sources; box and whisker plots (10–90% percentiles), lines at median (a). Changes in the concentration of cytokines secreted by dental pulp-derived naïve MSCs and mMSCs (48 h after the transfection); the values on the diagrams are the mean ± SD of three independent analyses, each of them being performed in triplicate (b). * p < 0.05 compared to the specified values.
Figure 5
Figure 5
A comparative analysis of the cellular immune response in mice to the HCV NS5A protein in vitro after immunization with naïve MSC, mMSC, and the plasmid. The four groups (Gr) of mice were injected three times with non-transfected MSC (Gr1), mMSC (Gr2), the pcNS5A-GFP plasmid (Gr3), or saline (Gr4). To assess the cellular response of lymphocytes in vitro, we used the peptides and recombinant proteins from the NS5A region which were combined into two pools; the medium alone was used as a negative control. The results of T cell proliferation are expressed as stimulation indexes (SIs) (a); the IFN-γ production by splenocytes in response to NS5A antigens was assayed as the number of IFN-γ-synthesizing cells by ELISpot in the number of spot-forming cells (SFC) per 106 cells (b). The values on each diagram are the mean ± SD of three measurements done in three independent experiments. * p < 0.05 compared to control; ** p < 0.05 compared to the specified groups.
Figure 6
Figure 6
A comparative analysis of the proportion of cells in the spleens of immunized mice. Splenocytes from the immunized mice were stained with anti-CD4 and anti-CD8 antibodies (a) with the Mouse Naïve/Memory T Cell Panel kit (b) or with anti-CD11c, anti-CD11b, and anti-Gr1 antibodies (c), and analyzed by multicolor flow cytometry. Representative dot plots and values of the number of CD4 and CD8-positive cells are shown as percentages (a); representative dot plots and values of the number of CD4+ naïve (CD62high/CD44low), effector (CD62low/CD44high), and memory (CD62high/CD44high) T cells subsets are shown as percentages (b); representative dot plots and values of the number of MDSC expressing CD11b and Gr1, and negative for the marker of DC (CD11c) are shown as percentages (c); mean values of CD4+/CD8+ ratio, CD4+ T-memory cells, and MDSC are shown as percentages (d). The values on the diagram are the mean ± SD of three independent analyses, and each of them was performed in triplicate. * p < 0.05 compared to the control Gr4.
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