Staphylococcal Enterotoxin B Challenge Research Articles

Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine potential in a mouse model

Superantigens produced by Staphylococcus aureus can cause food poisoning and toxic shock syndrome. The biological activities and vaccine potential of mutant staphylococcal enterotoxin B (SEB) proteins, N23K and F44S, were studied in a lipopolysaccharide-potentiated mouse model. Although 10 μg of SEB per mouse is equivalent to 30 LD 50, the same intraperitoneal dose of either mutant protein was nonlethal and did not elevate serum levels of tumor necrosis factors (TNF). N23K, F44S, and SEB were serologically identical in an enzyme-linked immunosorbent assay with polyclonal anti-SEB. Immunization with alum containing N23K, F44S, or SEB elicited an anti-SEB response that protected 80–87% of the mice against a 10 μg SEB challenge. Controls lacking an anti-SEB titer did not survive. Pooled sera from immunized mice effectively blocked SEB-induced T-cell proliferation in vitro. Naive mice survived a lethal SEB challenge when given pooled antisera 1, 2, or 4 h later, whereas the antisera failed to protect animals when administered 6 or 8 h after the toxin. Lethality at the later times was consistent with increased serum levels of TNF observed 6 h after SEB injection. These studies suggest that the N23K and F44S mutant proteins of SEB are less biologically active than the wild-type toxin, yet retain epitopes useful for eliciting a protective antibody response.

Apoptosis of crypt cells and inflammatory reactions in the small intestine of mice challenged with staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB), a superantigen that stimulates activation and proliferation of T cells bearing certain Vb elements of the T cell receptor, causes food poisoning and toxic shock in humans and in monkeys. The clinical symptoms of SEB toxicosis include nausea, vomiting, retching, abdominal cramping, diarrhea, headache, muscular cramping and marked prostration with a considerable drop in blood pressure in some cases. The mechanism by which SEB causes these symptoms is still obscure. Production of large amounts of cytokines following SEB challenge was assumed to lead to toxic shock. We have recently developed a SEB toxic shock model in mice by priming with actinomycin D (Act-D). Mice primed with Act-D become very sensitive to SEB-induced toxicosis and death. Enhancement of cytokine expression in the spleen and leukocyte sequestration in the lung as well as increased adhesive molecule expression were reported previously. This report describes the pathological reactions found in the small intestine and discusses the pathogenesis of those abnormalities.KeywordsSmall IntestineCrypt CellStaphylococcal EnterotoxinCytokine Release SyndromeWalter Reed Army InstituteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Hypothalamic-pituitary-adrenal activation by the bacterial superantigen staphylococcal enterotoxin B: role of macrophages and T cells.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen which stimulates T cells bearing the V beta 8 motif on the T-cell receptor. This stimulation is MHC class II dependent, and in vivo results in a rapid and pronounced T-cell cytokine response. Based on previous evidence that SEB stimulates corticosterone production in BALB/c mice, which possess a high percentage of V beta 8+ T cells, we explored the effects of SEB on the hypothalamic-pituitary-adrenal (HPA) axis and identified the peripheral immunologic cellular requirements for these effects. Administration of SEB stimulates corticosterone in a dose-dependent manner, with peak production of corticosterone occurring by 2 h after intraperitoneal challenge with 50 micrograms SEB. Challenge with staphylococcal enterotoxin A, which activates V beta 3+ and V beta 11+ T cells (deleted during ontogenesis in BALB/c mice), did not increase ACTH or corticosterone production. Furthermore, SEB challenge increased plasma ACTH, which accounted for the increased plasma corticosterone, and increased the expression of c-fos in the PVN region of the hypothalamus. In vivo elimination of macrophages did not prevent the corticosterone response to SEB, suggesting that pituitary-adrenal activation does not require macrophages. However, when mice were pretreated with the T-cell immunosuppressant cyclosporin A, the significantly increased ACTH and corticosterone production in response to SEB was dramatically attenuated. These results demonstrate that bacterial superantigens can stimulate the HPA axis, and that functional T cells may play an obligatory role in this effect.

Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines.

Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound [correction of would] infections or viral influenza-associated staphylococcal superinfections or when aerosolized for use as a potential biologic warfare threat agent. Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated with meningococcal outer membrane protein proteosomes has previously been shown to be immunogenic and protective against lethal respiratory or parenteral SEB challenge in murine models of SEB intoxication. Here, it is demonstrated that immunization of nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10 per group) were primed i.m. and given booster injections by either the i.m. or intratracheal route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG) responses were elicited in all monkeys, but strong IgA responses in sera and bronchial secretions were elicited both pre- and post-SEB challenge only in monkeys given booster injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both routes in protecting 100% of monkeys against severe symptomatology and death from aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and enhancement of both bronchial and systemic IgA and IgG responses by the proteosome vaccine delivered by a respiratory route are also encouraging for the development of mucosally delivered proteosome vaccines to protect against SEB and other toxic or infectious respiratory pathogens.

Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice

Intranasal or intramuscular (i.m.) immunization of mice and i.m. immunization of rabbits with formalinized staphylococcal enterotoxin B (SEB) toxoid in saline elicited higher anti-SEB serum immunoglobulin G (IgG) titers when the toxoid was formulated with proteosomes. In addition, intranasal immunization of mice with this proteosome-toxoid vaccine elicited high levels of anti-SEB IgA in lung and intestinal secretions, whereas the toxoid without proteosomes did not. Two i.m. immunizations with proteosome-toxoid plus alum also induced higher murine serum responses than alum-adjuvanted toxoid without proteosomes. Furthermore, proteosome-toxoid delivered intranasally in saline or i.m. with either saline or alum afforded significant protection against lethal SEB challenge in two D-galactosamine-sensitized murine models of SEB intoxication, i.e., the previously described i.m. challenge model and a new respiratory challenge model of mucosal SEB exposure. Efficacy correlated with the induction of high serum levels of anti-SEB IgG. In contrast, intranasal or i.m. immunization with toxoid in saline without proteosomes was not significantly protective in either challenge model. Proteosome-toxoid plus alum given i.m. also elicited more significant protection against respiratory challenge than the alum-adjuvanted toxoid alone. The capacity of proteosomes to enhance both i.m. and intranasal immunogenicity and efficacy of SEB toxoid indicates that testing such proteosome-SEB toxoid vaccines in the nonhuman primate aerosol challenge model of SEB intoxication prior to immunogenicity trials in humans is warranted. These data expand the applicability of the proteosome mucosal vaccine delivery system to protein toxoids and suggest that respiratory delivery of proteosome vaccines may be practical for enhancement of both mucosal and systemic immunity against toxic or infectious diseases.

Humoral immunity to aerosolized staphylococcal enterotoxin B (SEB), a superantigen, in monkeys vaccinated with SEB toxoid-containing microspheres.

Staphylococcal enterotoxin B (SEB) toxoid-containing microspheres were tested for efficacy in rhesus monkeys as a vaccine candidate for respiratory SEB toxicosis and toxic shock. Forty monkeys were randomly separated into 10 groups of four monkeys each: 9 groups were vaccinated with the microspheres via combinations of mucosal and nonmucosal routes, and 1 group served as nonvaccinated controls. Both vaccinated and nonvaccinated monkeys were then challenged with a high lethal dose of SEB aerosol. Monkeys primed with an intramuscular dose of the microspheres followed by an intratracheal booster all survived the SEB challenge. Overall, monkeys with an intratracheal booster generally had the highest antibody levels, which is consistent with their high survival rate and lower rate of illness. Protective immunity was correlated with antibody levels in both the circulation and the respiratory tract. The protection was not due to the depletion or anergy of SEB-reactive T cells, since SEB-induced proliferation in cultures of circulating lymphocytes was not significantly reduced after the microsphere vaccination. It is evident that the nonsurvivors did not die of systemic anaphylaxis or hypersensitivity because the monkeys did not die immediately after SEB challenge and there were no significant differences in histamine levels between the vaccinated and control monkeys before and after SEB challenge. The antibodies seemed to neutralize the SEB that got into the airway and the circulation.

The protective role of endogenously synthesized nitric oxide in staphylococcal enterotoxin B-induced shock in mice.

Nitric oxide (NO) synthesis during experimental endotoxemia has been shown to have both deleterious and beneficial effects. In the present study, we analyzed the in vivo production and the regulatory role of NO in the shock syndrome induced by staphylococcal enterotoxin B (SEB) in mice. First, we found that intraperitoneal administration of 100 micrograms SEB in BALB/c mice induced a massive synthesis of NO as indicated by high serum levels of nitrite (NO2-) and nitrate (NO3-) peaking 16 h after SEB injection. The inhibition of NO2- and NO3- release in mice injected with anti-tumor necrosis factor (TNF) and/or anti-interferon gamma (IFN-gamma) monoclonal antibody (mAb) before SEB challenge revealed that both cytokines were involved in SEB-induced NO overproduction. In vitro experiments indicated that NO synthase (NOS) inhibition by N-nitro-L-arginine methyl ester (L-NAME) enhanced IFN-gamma and TNF production by splenocytes in response to SEB. A similar effect was observed in vivo as treatment of mice with L-NAME resulted in increased IFN-gamma and TNF serum levels 24 h after SEB challenge, together with persistent expression of corresponding cytokine mRNA in spleen. The prolonged production of inflammatory cytokines in mice receiving L-NAME and SEB was associated with a 95% mortality rate within 96 h, whereas all mice survived injections of SEB or L-NAME alone. Both TNF and INF-gamma were responsible for the lethality induced by SEB in L-NAME-treated mice as shown by the protection provided by simultaneous administration of anti-IFN-gamma and anti-TNF mAbs. We conclude the SEB induces NO synthesis in vivo and that endogenous NO has protective effects in this model of T cell-dependent shock by downregulating IFN-gamma and TNF production.

Immunity and responses of circulating leukocytes and lymphocytes in monkeys to aerosolized staphylococcal enterotoxin B

Rhesus monkeys immunized intramuscularly or orally with staphylococcal enterotoxin B (SEB) toxoid or SEB toxoid incorporated in microspheres made of poly(DL-lactide-co-glycolide) were challenged with a lethal dose of aerosolized SEB to study their immunity and cellular responses in the circulation. It was found that circulating antibodies play a critical role in preventing SEB from triggering toxicosis. Monkeys with high levels of antibodies survived, while those with low levels underwent 2 to 3 days of toxicosis and died. Intramuscular immunization induced high levels and oral immunization induced low levels of antibodies. The circulating antibodies in surviving monkeys decreased dramatically within 20 min and started to rebound at 90 min after SEB challenge. At 90 min, the dying monkeys showed in the circulation a dramatic increase of polymorphonuclear leukocytes and decreases of NK cells and monocytes (CD16 and CD56 markers) as well as of lymphocytes with HLA-DR, CD2, CD8, and IL2R alpha (CD25) markers. The number of polymorphonuclear leukocytes showed an inverse correlation with the numbers of monocytes and various lymphocyte subpopulations which, except for IL-2R, CD16, and CD56(+) cells, showed a direct correlation with one another. The changes in the populations of leukocytes, monocytes, NK cells, and lymphocytes seem to be an indication of initial toxicosis; however, the roles of these cells in toxicosis and death remain to be defined.

Defective maintenance of T cell tolerance to a superantigen in MRL-lpr/lpr mice.

In normal mice neonatal injection of staphylococcal enterotoxin B (SEB) induces tolerance in T cells that express reactive T cell receptor (TCR) V beta regions. To determine if a T cell neonatal defect was present in MRL-lpr/lpr mice, 20 micrograms of SEB was injected intraperitoneally every other day into V beta 8.2 TCR transgenic and nontransgenic MRL(-)+/+ and MRL-lpr/lpr mice from birth to 2 wk of age. At 2 wk of age, V beta 8+ T cells were depleted, and SEB reactivity was lost, in spleen, lymph node, and thymus. These effects were equivalent in +/+ and lpr/lpr SEB-tolerized mice. However, MRL-lpr/lpr mice failed to maintain neonatal tolerance. By 4 wk of age, there was a dramatic increase in T cells expressing V beta 8.2 in the peripheral lymph nodes of MRL-lpr/lpr mice but not MRL(-)+/+ mice. In vitro stimulation with SEB or TCR crosslinking revealed a total loss of neonatal tolerance 2 wk after cessation of SEB treatment in lpr/lpr mice, but not +/+ mice. The time-course of recovery of V beta 8+ T cells and reactivity to SEB and TCR crosslinking in the thymus of MRL-lpr/lpr mice was similar to that in the lymph node. Thymectomy at 2 wk of age eliminated tolerance loss in lymph nodes of MRL-lpr/lpr mice at 4 wk of age, indicating that loss of peripheral tolerance was due to the emigration of untolerized T cells from the thymus. Challenge of neonatally tolerized MRL-lpr/lpr mice with SEB (100 micrograms, i.p.) at 8 wk of age resulted in a dramatic onset of T cell-mediated autoimmune disease characterized by 30% weight loss and 60% morality. This indicated that loss of tolerance to SEB also occurred in vivo. In contrast, neonatally tolerized MRL(-)+/+ mice remained totally unresponsive to SEB challenge and did not undergo any detectable weight loss. These results suggest that there is normal induction of neonatal tolerance to SEB in lpr/lpr mice, but that tolerance is not maintained after the tolerizing antigen is removed. This loss of neonatal tolerance can lead to severe weight loss and death on exposure to the tolerizing antigen later in life.

Role of substance P in immediate-type skin reactions induced by staphylococcal enterotoxin B in unsensitized monkeys

The staphylococcal enterotoxin B (SEB)-induced immediate-type skin reaction in unsensitized monkeys was used as a nonimmunologic mast cell stimulation to search for possible involvement of local neural mechanisms. Evidence is presented that substance P (SP) plays a predominant role in mediating intradermal SEB challenge in unsensitized monkeys. With a rabbit SP antiserum directed against the C-terminal region of SP, a concentration-dependent inhibition of SEB-induced skin reactivity could be demonstrated. Furthermore, a rabbit antiserum directed against the mast cell activating N-terminal part of SP was capable of impeding SEB-induced skin reactions totally. By use of SP antagonists, significant reduction of skin reactions evoked by SEB was found. Finally, capsaicin pretreatment of the skin caused a substantial inhibition of SEB-induced skin reactivity. These data suggest that SEB exerts its effect on cutaneous mast cells via stimulation of primary sensory neurons that contain SP. Moreover, a new in vivo model is described for studies of nerve-mast cell interactions.

Staphylococcal enterotoxin B as a nonimmunological mast cell stimulus in primates: the role of endogenous cysteinyl leukotrienes.

The immediate-type skin reaction and the emetic response in unsensitized monkeys on challenge with staphylococcal enterotoxin B (SEB) were studied to define the role of cysteinyl leukotrienes (LTs) in the action of the toxin. LY 171883, a selective LTD4/LTE4 receptor inhibitor, antagonized SEB-induced skin reactions and emetic responses completely. Inhibition of prostanoid formation by indomethacin, however, and pretreatment with BW 755C, a dual lipoxygenase and cyclooxygenase inhibitor, did not influence these reactions. The generation of endogenous cysteinyl LTs upon intragastric SEB administration was established in vivo. There was a tenfold increase in LTE4, the major biliary cysteinyl LT, and a novel cysteinyl LT metabolite in urine occurred, indicating strongly enhanced LT generation on SEB challenge. These results provide the first evidence that cysteinyl LTs may be important mediators in the pathophysiology of SEB-induced effects, as a model for pseudo-allergic reactions.