Wild-type Staphylococcal Enterotoxin B Research Articles

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

Oral administration of recombinant Bacillus subtilis spores expressing mutant staphylococcal enterotoxin B provides potent protection against lethal enterotoxin challenge

Pathogenicity of Staphylococcus aureus is induced by staphylococcal enterotoxin B (SEB). A mutant form of SEB (mSEB) is immunogenic as well as less toxic. Recombinant mSEB and SEB were expressed in pET28a prokaryotic plasmids. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in mSEB-stimulated macrophages were lower than those in SEB-stimulated macrophages (p < 0.001, p < 0.01 respectively). Using CotC as a fusion protein, we constructed recombinant Bacillus subtilis spores expressing mSEB on the spore surface and evaluated their safety and protective efficacy via mouse models. Oral administration of mSEB-expressing spores increased SEB-specific IgA in feces and SEB-specific IgG1 and IgG2a in the sera, compared with mice in naïve and CotC spore-treated groups (p < 0.001, p < 0.01, p < 0.001 respectively). Six weeks following oral dosing of recombinant spores, significant differences were not found in the serum biochemical indices between the mSEB group and the naïve and CotC groups. Furthermore, oral administration of mSEB spores increased the survival rate by 33.3% in mice intraperitoneally injected with 5 µg of wild-type SEB plus 25 µg lipopolysaccharide (LPS). In summation, recombinant spores stably expressing mSEB were developed, and oral administration of such recombinant spores induced a humoral immune response and provided protection against SEB challenge in mice.

Structure of the staphylococcal enterotoxin B vaccine candidate S19 showing eliminated superantigen activity.

Four mutations (N23A, Y90A, R110A and F177A) were introduced into S19, a vaccine candidate for staphylococcal enterotoxin B (SEB), resulting in a lower binding affinity towards the T-cell receptor beta chain (TCB) and reducing its superantigen activity. The structure of S19 was solved and was superposed on the native or complex structure of SEB. In the superposition model, mutations that were introduced seemed to reduce the number of hydrogen bonds at the SEB-TCB interface. S19 also displayed an unexpected structural change around the flexible-loop region owing to the Y90A mutation. This local structural change provided evidence that the mutated form of S19 could have a lower affinity for major histocompatibility complex (MHC) class II than wild-type SEB.

Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue

The nasopharynx-associated lymphoid tissue (NALT) of humans and other mammals is associated with immunity against airborne infections, though it is generally considered to be a secondary component of the mucosa-associated lymphoid system. We found that protective immunity to a virulence factor of nasal mucosa-colonizing Staphylococcus aureus, staphylococcal enterotoxin B (SEB), requires a functional NALT. We examined the role of NALT using intranasal (IN) vaccination with a recombinant SEB vaccine (rSEBv) combined with an adjuvant in a mouse model of SEB-induced toxic shock. The rSEBv was rapidly internalized by NALT cells at the mucosal barrier, and transport into NALT was accelerated by inclusion of a Toll-like receptor 4 (TLR4) agonist. Vaccine-induced germinal centers of B cells formed within NALT, accompanied by elevated levels of IgA(+) and IgG(+) cells, and these were further increased by TLR4 activation. The NALT was the site of specific anti-rSEBv IgA and IgG production but was also influenced by intraperitoneal (IP) inoculation and perhaps other isolated lymphoid follicles observed within the nasal cavity. Vaccination by the IN route generated robust levels of anti-rSEBv IgA in saliva, nasal secretions, and blood compared to much lower levels after IP vaccination. IN vaccination also induced secretion of anti-rSEBv IgG in the blood and nasal secretions. Significantly, the efficacy of IN vaccination was dependent on NALT, as surgical removal resulted in greater sensitivity to IN challenge with wild-type SEB. Thus, protective immunity to SEB within the nasal sinuses was elicited by responses originating in NALT.

Contribution of the flexible loop region to the function of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB), a toxin produced by Staphylococcus aureus, causes food poisoning and other fatal diseases by inducing high levels of pro-inflammatory cytokines. These cytokines are released from CD4+ T cells and major histocompatibility complex (MHC) class II antigen-presenting cells, which are activated through binding of wild-type (WT) SEB to both the MHC class II molecule and specific T-cell receptor Vbeta chains. Here, we focused on a trypsin/cathepsin cleavage site of WT SEB, which is known to be cleaved in vivo between Lys97 and Lys98, located within the loop region. To know the function of the cleavage, an SEB mutant, in which both of these Lys residues have been changed to Ser, was examined. This mutant showed prolonged tolerance to protease cleavage at a different site between Thr107 and Asp108, and structural analyses revealed no major conformational differences between WT SEB and the mutant protein. However, differential scanning calorimetric analysis showed an increase in enthalpy upon thermal denaturation of the mutant protein, which correlated with the speed of cleavage between Thr107 and Asp108. The mutant protein also had slightly increased affinity for MHC. In the in vivo experiment, the SEB mutant showed lower proliferative response in peripheral blood mononuclear cells and had lower cytokine-induction activity, compared with WT SEB. These results highlight the importance of the flexible loop region for the functional, physical and chemical properties of WT SEB, thus providing insight into the nature of WT SEB that was unrevealed previously.

Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity

At this time there are no vaccines or therapeutics to protect against staphylococcal enterotoxin B (SEB) exposure. Here, we report vaccine efficacy of an attenuated SEB in a nonhuman primate model following lethal aerosol challenge and identify several biomarkers of protective immunity. Initial in vitro results indicated that the mutation of key amino acid residues in the major histocompatibility complex (MHC) class II binding sites of SEB produced a nontoxic form of SEB, which had little to no detectable binding to MHC class II molecules, and lacked T-cell stimulatory activities. When examined in a mouse model, we found that the attenuated SEB retained antigenic structures and elicited protective immune responses against wild-type SEB challenge. Subsequently, a vaccine regimen against SEB in a nonhuman primate model was partially optimized, and investigations of immune biomarkers as indicators of protection were performed. SEB-naïve rhesus monkeys were vaccinated two or three times with 5 or 20 μg of the attenuated SEB and challenged by aerosol with wild-type SEB toxin. Unlike exposure to the native toxin, the vaccine did not trigger the release of inflammatory cytokines (TNFα, IL6, or IFNγ). All rhesus monkeys that developed anti-SEB serum titers ≥10 4 and elicited high levels of neutralizing antibody survived the aerosol challenge. These findings suggest that the attenuated SEB is fully protective against aerosolized toxin when administered to unprimed subjects. Moreover, experiments presented in this study identified various biomarkers that showed substantial promise as correlates of immunity and surrogate endpoints for assessing in vivo biological responses in primates, and possibly in humans, to vaccines against SEs.

Toxicity of the staphylococcal enterotoxin B mutants with histidine-to-tyrosine substitutions

In this study we made a series of site-directed mutants of staphylococcal enterotoxin B (SEB), in which histidine residues in the molecule were replaced by tyrosine. The mutant genes were cloned and expressed, and the corresponding proteins were purified. These mutant proteins were tested for binding to human HLA-DR4 and for mitogenetic activity in mouse splenocyte culture. Toxicity of the proteins in vivo was evaluated in the actinomycin D-primed C3H/HeJ mouse model. We found that SEB mutant proteins with fewer than four histidine-to-tyrosine (his-to-tyr) substitutions retained toxic properties similar to wild-type SEB. However, studies showed that his-to-tyr substitution of four consecutive histidine residues eliminated SEB toxicity. Our results clearly show that this genetically modified SEB protein is non-toxic and justifies its further development as a component of a new, safer vaccine to prevent SEB intoxication.

Functional analysis of Mycoplasma arthritidis-derived mitogen interactions with class II molecules.

The ability of superantigens (SAGs) to trigger various cellular events via major histocompatibility complex (MHC) class II molecules is largely mediated by their mode of interaction. Having two MHC class II binding sites, staphylococcal enterotoxin A (SEA) is able to dimerize MHC class II molecules on the cell surface and consequently induces cytokine gene expression in human monocytes. In contrast, cross-linking with specific monoclonal antibodies or T-cell receptor is required for staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1) to induce similar responses. In the present study, we report how Mycoplasma arthritidis-derived mitogen (MAM) may interact with MHC class II molecules to induce cytokine gene expression in human monocytes. The data presented indicate that MAM-induced cytokine gene expression in human monocytes is Zn2+ dependent. The MAM-induced response is completely abolished by pretreatment with SEA mutants that have lost their capacity to bind either the MHC class II alpha or beta chain, with wild-type SEB, or with wild-type TSST-1, suggesting that MAM induces cytokine gene expression most probably by inducing dimerization of class II molecules. In addition, it seems that SEA and MAM interact with the same or overlapping binding sites on the MHC class II beta chain and, on the other hand, that they bind to the alpha chain most probably through the regions that are involved in SEB and TSST-1 binding.

Variable influence of MHC polymorphism on the recognition of bacterial superantigens by T cells.

Superantigen recognition by some T cells is strongly influenced by polymorphic residues of the MHC class II molecule, suggesting that the TCR contacts the class II molecule during superantigen engagement. However, the degree of MHC preference varies with V beta expression. For example, the recognition of staphylococcal enterotoxin B (SEB) by murine V beta 14+ T cell hybridomas is strongly influenced by MHC polymorphism, whereas the recognition of SEB by V beta 8.2+ hybridomas appears to be independent of MHC polymorphism. One possible explanation for this difference is that V beta 14+ TCR may have a lower avidity for SEB such that the stability of the TCR/SEB/MHC complex is more strongly influenced by potential TCR/MHC interactions. To investigate this possibility, we examined the MHC preference of SEB recognition by murine V beta 8.2+/CD4+ T cell hybridomas in which we altered the strength of SEB recognition. First, we compared the recognition of wild-type SEB and mutant SEB, which only weakly activates T cells. Second, we compared recognition of SEB presented by I-E and I-A molecules, which differ in their affinity for SEB. In both cases, the degree of MHC preference was significantly increased when the strength of SEB recognition was reduced. Furthermore, we used mutant MHC class II molecules to show that the degree of MHC preference was controlled by residues of the class II molecule predicted to interact with the TCR during SEB recognition. Taken together, these data support the idea that there is a TCR/MHC interaction during SEB recognition, and the influence of this interaction varies with the overall avidity of the TCR/SEB/MHC complex.