Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects 15 to 30% of children and ~5% of adults in industrialized countries1. Although the pathogenesis of AD is not fully understood, the disease is mediated by an abnormal immunoglobulin E (IgE) immune response in the setting of skin barrier dysfunction2. Mast cells (MCs) contribute to IgE-mediated allergic disorders including AD3. Upon activation, MCs release their membrane-bound cytosolic granules leading to the release of multiple molecules that are important in the pathogenesis of AD and host defense4. More than 90% of AD patients are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbor the pathogen5. Several Staphylococcal exotoxins (SEs) can act as superantigens and/or antigens in models of AD6. However, the role of these SEs in disease pathogenesis remains unclear. Here, we report that culture supernatants of S. aureus contain potent MC degranulation activity. Biochemical analysis identified δ-toxin as the MC degranulation-inducing factor produced by S. aureus. MC degranulation induced by δ-toxin depended on phosphoinositide 3-kinase (PI3K) and calcium (Ca2+) influx, but unlike that mediated by IgE crosslinking, it did not require the spleen tyrosine kinase (Syk). In addition, IgE enhanced δ-toxin-induced MC degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from AD patients produced high levels of δ-toxin. Importantly, skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted IgE and IL-4 production, as well as inflammatory skin disease. Furthermore, enhancement of IgE production and dermatitis by δ-toxin was abrogated in KitW-sh/W-sh MC-deficient mice and restored by MC reconstitution. These studies identify δ-toxin as a potent inducer of MC degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2␤2-2␤3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region. Overall, four out of six rabbits vaccinated with the MAP peptide in Freund's adjuvant developed high-titer, high-avidity antibody responses which cross-reacted with the immobilized peptide sequence comprising aa 305 to 319 and with PA, as determined by an enzyme-linked immunosorbent assay, and which displayed potent and durable neutralization of lethal toxin (LeTx) in vitro, with peak titers which were 452%, 100%, 67%, and 41% of the peak neutralization titers observed in positive-control rabbits immunized with PA. Importantly, analysis of sera from multiple cohorts of rabbits with high-titer immunity to PA demonstrated a virtual absence of this potent antibody specificity, and work by others suggests that this specificity may be present at only low levels in primate PA antiserum. These results highlight the potential importance of this immunologically cryptic neutralizing epitope from PA as a target for alternative and adjunctive vaccines for anthrax.

We previously showed that a multiple antigenic peptide (MAP) displaying amino acids (aa) 305 to 319 from the 2␤2-2␤3 loop of protective antigen (PA) can elicit high-titered antibody that neutralizes lethal toxin (LeTx) in vitro and that this loop-neutralizing determinant (LND) specificity is absent in PA-immune rabbits. Some immune rabbits were, however, nonresponders to the MAP. We hypothesized that the immunogen elicited suboptimal major histocompatibility complex (MHC) class II-restricted T-cell help and that introduction of a functional helper T-cell epitope would increase MHC-restricted responsiveness and the magnitude and affinity of the antibody responses. In the current study, we characterized the T-and B-cell responses to LND peptides in mice, then designed second-generation MAP immunogens for eliciting LND-specific immunity, and tested them in rabbits. Bacillus anthracis is a gram-positive, spore-forming bacterium that naturally infects wildlife and livestock and, less frequently, humans. Since 2001, when spores of B. anthracis sent through the U.S mail resulted in infection in 22 individuals, including five fatal cases of inhalation anthrax, considerable effort has been directed toward reevaluating our preparedness for possible bioterrorist threats, including weaponized anthrax.The morbidity and mortality associated with inhalation anthrax are largely a result of the elaboration of two toxins, lethal toxin (LeTx) and edema toxin. These toxins are classic A-B toxins, where lethal factor and edema factor represent the active moieties and protective antigen (PA) represents the cell-binding moiety (5,6,20). Humoral immunity to PA can successfully mediate protection from lethal challenges in animal models of inhalation anthrax, and the protection is correlated with the ability of PA-specific antibodies to neutralize LeTx in vitro in the toxin neutralization assay (TNA) (17,21,22,35,36,41).While PA-specific antibody titer has been shown to correlate with TNA titers in vitro, this relationship demonstrates variability among different studies and in different species (19,37,40,43,48,50). Most PA-specific neutralizing monoclonal antibodies (MAbs) show a more invariable linear relationship between concentration and toxin neutralization (4, 46). These findings can be reconciled, in part, by the fact that only a fraction of the polyclonal antibody produced in response to vaccination with PA contributes to LeTx neutralization (4,39,40). Indeed, analyses of human and murine MAbs suggest that whereas immunization with whole PA elicits antibodies to a wide range of sequences within the protein, the repertoire of neutralizing antibodies is considerably more focused, limited perhaps to only a couple of major regions in PA, including the anthrax toxin receptor binding region in domain 4 and the lethal factor-and edema factor-binding regions in domain 1 (4,24,25,40). Though PA-specific antibody may contribute to protection through mechanisms other than neutralization, for example, by facilitating opsonization of spores (...