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The 56-kD outer membrane protein of Orientia tsutsugamushi has previously been shown to be the immunodominant antigen in scrub typhus infections. Its gene was cloned into the DNA vaccine vector pVR1012 as a vaccine candidate (pKarp56). The in vitro expression of this 56-kD antigen by pKarp56 was confirmed in tissue culture by an indirect fluorescence assay and Western blot analysis. The initial antibody responses of mice immunized with varied doses of the pKarp56 were barely detected, but increases were observed after each of three subsequent booster immunizations. Although no protection was observed with a single immunization of pKarp56, after four immunizations, 60% of the mice survived a 1,000 × 50% lethal dose (LD50) challenge. These results specifically confirm the importance of the 56-kD protein antigen in protective immunity against O. tsutsugamushi and demonstrate the feasibility of DNA vaccines for the prevention of scrub typhus.