DNA minigene vaccine pHI-FLK₄₀₀ suppresses tumor metastasis and the immunity induced by pHI-Db is long-lasting. (A) Surface expression of FLK-1 (black dotted line) or H-2D^b (black solid line) of EO771 breast carcinoma cells. Isotype control is shown with gray shaded curve. (B) Groups of vaccinated mice (n = 8) were challenged intravenously with 2 × 10⁵ EO771 breast carcinoma cells 2 weeks after the last vaccination. Mice were killed 21 days later and lung weights assessed. ^*P < .01 compared to pCMV and pHI; ^**P < .005 compared to pCMV and pHI; ^***P < .05, P < .05, P > .05, and P > .05 compared to pCMV, pHI, pHI-Db, and pHI-FLK₄₀₀, respectively. (C) Groups of vaccinated mice (n = 4) were challenged intravenously with 2 × 10⁵ EO771 breast carcinoma cells 10 months after the last vaccination. ^*P < .01 compared to the pCMV control group. (D) ELISPOT assay performed with splenocytes isolated from mice (n = 4) 10 months after pHI-Db vaccination in the presence or absence of FLK₄₀₀ peptide. ^*P < .02 compared to culture medium alone. Error bars indicate SD.