Fig. 4

M2 vaccine elicits a poly-functional and long lasting immune response. (a) Experimental set up: mice were vaccinated with the M2 vaccine by DNA-EP once every two weeks for three times and immune responses were analyzed on day 35 and 58. (b) The immune response was measured in the peripheral blood by FC on day 7 after the last vaccination. Paired matched T-test 2-tailed *p < 0.05, **p < 0.01, ***p < 0,001, ****p < 0,0001. (c) Splenocytes from the M2 vaccinated mice are activated by MC38 cells. Mice were vaccinated according to the scheme and splenocytes were collected on day 35. Splenocytes from mice treated with a different vaccine control (pTK1) or M2 (upper row) were incubated with diverse stimulating cells (lower row) MC38 cells (MC38), with MC38 cells transfected with the M2 vaccine (MC38-M2) or MC38 cells transfected with an unrelated vaccine (MC38-C). The first group on the left represent the background signal that is given by splenocytes from mice vaccinated with control plasmid and stimulated with MC38 (Control/MC38). Dots represent IFN-γ production of splenocytes from single mice measured by FC, median and SD, *p < 0.05, **p < 0.01 Mann-Whitney test. (d)The analysis of immune responses was performed on day 7 (D35) and on day 30 (D58) after the last vaccination in the peripheral blood by ICS. Dots represent values of individual mice of two independent experiments with six-seven mice per group