Our results demonstrated that once administered orally, rmSEB remained sufficiently intact to stimulate a systemic (Fig.4A) and mucosal (Fig.4B) antibody response. be a safe subunit vaccine. To test this possibility, piglets Inolitazone 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 Inolitazone in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion Inolitazone 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 aureusproduces several exotoxins that are important determinants of pathogenicity (7). The staphylococcal enterotoxins are among these exotoxins and are produced byS. aureusstrains 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 CAPN2 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. aureuscan produce SEB within the environment, but its production is most problematic following contamination or when present in contaminated foodstuffs. Ingestion of the toxin results in symptoms which include anorexia, nausea, vomiting, and diarrhea, which may Inolitazone 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 a very compact, stable protein structure, allowing it to survive the harsh environment of the gastrointestinal tract (24,31). In addition, its stability to heat and denaturation allowed the weaponization of this toxin for aerosol dispersal in the 1960s (4). Following inhalation of aerosolized SEB, patients experience shortness of breath, chest pain, and some tachycardia (26). If exposure is significant, pulmonary edema, high fever, and a respiratory distress-like syndrome occur. With supportive medical intervention, death following inhalation is not common. However, symptoms and incapacitation can linger for up to 2 weeks following exposure. SEB has been characterized as one of the two most important toxin threats around the battlefield or in bioterrorism (17). At present, there are no approved vaccines for SEB. Early attempts at toxoid-based formalin-inactivated vaccines have been forgotten, since these toxoids were not reproducibly protective (33). This has led to more recent investigations using engineered, nontoxic mutant forms of SEB. These mutant forms of SEB have been designed and tested based on the considerable data defining the structure-function relationships for this toxin (13,22,23). Specifically, several studies have focused on the role of particular amino acid residues in SEB that are important in toxinogenic activity (1,3,5,15,24,32,34). Most staphylococcal superantigens have common structures for binding to a subunit of the human MHC class II molecule (34). A hydrophobic binding loop, centered at a leucine residue (e.g., staphylococcal enterotoxin A [SEA] L48, SEB L45, and toxic shock syndrome toxin 1 [TSST-1] L30), is conserved in all superantigens except streptococcal pyrogenic exotoxin C and is essential for the recognition of the class II molecule. A second conserved.