Figure 3.

MHV nsp14 N7-MTase mutants induced both humoral and cellular immune responses in mice. (A) Scheme of immunization. Four-week-old C57BL/6 mice were s.c. immunized with 5×10⁵ PFU N7-MTase mutants or WT viruses. Sera were collected at 2 and 4 wpi. At 4 wpi, mouse spleens were harvested. (B) Virus-specific IgG titre in mice serum. Sera were 5-fold serial-diluted and the IgG titre was then measured by ELISA. Means with SEM are shown (n = 5). (C) Neutralizing antibody titre in sera of mice. To neutralize the antibody titre, sera were 2-fold serial-diluted to PRNT₅₀ test. Means with SEM are shown (n = 5). (D) Virus-specific neutralizing antibodies can exist for at least 48 weeks in the sera of immunized animals. Means with SEM are shown (n = 3∼5). (E) Spleens of immunized mice were collected at 4 wpi. Single splenocytes were cultured ex vivo and stimulated with WT MHV for 24 h, after which they were stained for markers (IFN-γ, CD3, CD4 or CD8). The T-cells were gated and the percentages of (F) IFN-γ⁺ CD4⁺ cells and (G) IFN-γ⁺ CD8⁺ cells are shown. Means with SEM are shown (n = 5). (G), (H), (I), and (J) Total splenocyte supernatant from the ex vivo culture was collected 48 h after WT MHV stimulation, and IFN-γ, IL-2, IL-10, and IL-4 production were measured by ELISA. Data are presented as means ± S.E.M. Means with SEM are shown (n = 5). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant, P > 0.05 (unpaired Student’s t-test).