In previous trials, live invasive Escherichia coli-Shigella flexneri 2a hybrid vaccine candidate EcSf2a-2, administered to adult volunteers as 3 doses of ca. 2 x 10(9) colony forming units (c.f.u.) spaced over one week, induced fever and/or diarrhea in 11% of subjects and provided only limited protection (36% efficacy) against illness following challenge with virulent S. flexneri 2a. We sought to improve the clinical safety of this vaccine by administering a lower inoculum, and to enhance protective immunity by administering additional booster doses at 2 weeks. Twenty-one healthy adults were immunized with ca. 7 x 10(8) c.f.u. of EcSf2a-2 on days 0, 3, 14, and 17. The vaccine consistently colonized the intestine without causing serious adverse reactions; mild diarrhea developed in one subject and low grade fever in another. Vaccination elicited an antibody secreting cell (ASC) response to lipopolysaccharide (LPS) in all subjects, which was highest on day 7 and notably diminished thereafter on days 10, 16, 21, and 24, suggesting that active mucosal immunity developed rapidly. The magnitude of the response was modest (geometric mean peak = 16 IgA ASC/10(6) peripheral blood mononuclear cells) and an IgG serological response to LPS was detected in only 19% of subjects. Following experimental challenge with virulent S. flexneri 2a administered with bicarbonate buffer, shigellosis (diarrhea, dysentery, or fever) developed in 10 of 16 vaccine recipients (63%) and in 12 of 14 unvaccinated controls (86%), resulting in a vaccine efficacy of 27% (95% confidence limits -197, 82, p = 0.15, 1-tailed).(ABSTRACT TRUNCATED AT 250 WORDS)