Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.Staphylococcus aureus produces several exotoxins that are important determinants of pathogenicity (7). The staphylococcal enterotoxins are among these exotoxins and are produced by S. aureus strains growing in contaminated food, with staphylococcal enterotoxin B (SEB) being the most potent of the exotoxins. SEB mediates its toxicity by linking major histocompatibility complex (MHC) class II molecules with T-cell receptors outside the antigen binding site (24). Several families of T lymphocytes expressing certain V beta T-cell receptors can be stimulated by this toxin, which can include up to 20% of the total T-cell population. The term "superantigen" has been given to SEB and similar toxins which have this ability to bridge MHC class II molecules and T-cell receptors, stimulating a large percentage of T lymphocytes in this unconventional manner (12). One result of this toxin-induced T-lymphocyte activation is the overproduction of certain cytokines which contribute to the clinical symptoms of SEB-induced toxicity and shock (8). S. aureus can produce SEB within the environment, but its production is most problematic following infection or when present in contaminated foodstuffs. Ingestion of the toxin results in symptoms which include anorexia, nausea, vomiting, and diarrhea, which may present with hypotension, tachycardia, and hyperperistalsis (18).Unfortunately, SEB has several characteristics which make it a candidate for possible use as an agent of biowarfare or bioterrorism. SEB has...