Staphylococcal Enterotoxin B Challenge Research Articles

A highly neutralizing human monoclonal antibody targeting a novel linear epitope on staphylococcal enterotoxin B

ABSTRACT Staphylococcal Enterotoxin B (SEB), produced by Staphylococcus aureus (S. aureus), is a powerful superantigen that induces severe immune disruption and toxic shock syndrome (TSS) upon binding to MHC-II and TCR. Despite its significant impact on the pathogenesis of S. aureus, there are currently no specific therapeutic interventions available to counteract the mechanism of action exerted by this toxin. In this study, we have identified a human monoclonal antibody, named Hm0487, that specifically targets SEB by single-cell sequencing using PBMCs isolated from volunteers enrolled in a phase I clinical trial of the five-antigen S. aureus vaccine. X-ray crystallography studies revealed that Hm0487 exhibits high affinity for a linear B cell epitope in SEB (SEB138–147), which is located distantly from the site involved in the formation of the MHC-SEB-TCR ternary complex. Furthermore, in vitro studies demonstrated that Hm0487 significantly impacts the interaction of SEB with both receptors and the binding to immune cells, probably due to an allosteric effect on SEB rather than competing with receptors for binding sites. Moreover, both in vitro and in vivo studies validated that Hm0487 displayed efficient neutralizing efficacy in models of lethal shock and sepsis induced by either SEB or bacterial challenge. Our findings unveil an alternative mechanism for neutralizing the pathogenesis of SEB by Hm0487, and this antibody provides a novel strategy for mitigating both SEB-induced toxicity and S. aureus infection.

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.

Epigenetic regulation of C-Kitlo FcɛR1a+ mast cells by cannabidiol during staphylococcal enterotoxin B-induced liver injury

Abstract Although recent studies have established cannabidiol (CBD) as a potent anti-inflammatory agent, the mechanisms governing these processes remain unclear. Our previous research demonstrated that CBD treatment induced expansion of CD11b+Gr-1+MDSC in the peritoneum, which suppressed T cell functions leading to attenuation of staphylococcal enterotoxin B (SEB)-mediated liver inflammation in mice. Using the same model, we characterize the effects of CBD administration in the liver. Briefly, mice were injected intraperitoneally (I.P.) with 50mg/kg of CBD for 4 days (d), followed by SEB challenge on d 3. Flow cytometry analysis revealed a significant reduction in Vβ8+ CD8+ T cells. In agreement with our earlier findings, mice treated with CBD displayed robust expansion of Gr-1+ MDSC in the liver, which was primarily comprised of Ly6CHI MHCIILO leukocytes. As mast cells have been found to play a role in CBD-mediated induction of MDSC via increased peroxisome proliferator-activated receptor gamma (PPARγ) signaling, we examined changes to liver mast cell populations. We discovered two distinct subsets of mast cells, C-KithiFcɛR1a+ and C-Kitlo FcɛR1a+present in the liver. CBD treatment promoted expansion of the C-Kitlo FcɛR1a+ subset. Analysis by miRNA microarray displayed enrichment of miRNAs targeting mast cell growth factor, KIT ligand (KITLG), and significant downregulation of miR-217-5p, which is conserved for the co-activator of PPARγ (PPARγC1A). Increased gene expression of PPARγ was confirmed by single-cell RNA sequencing (sc-RNAseq). Taken together, our results identify CBD induced epigenetic reduction of C-Kit expression and KITLG signaling in mast cells to favor PPARγ signaling as a novel anti-inflammatory mechanism.

Inhibition of miRNA-222-3p Relieves Staphylococcal Enterotoxin B-Induced Liver Inflammatory Injury by Upregulating Suppressors of Cytokine Signaling 1.

PurposeStaphylococcal enterotoxin B (SEB) has been well-documented to induce liver injury. miRNA-222-3p (miR-222-3p) was implicated in SEB-induced lung injury and several liver injuries. This study aimed to explore the role of miR-222-3p in SEB-induced liver injury.Materials and MethodsExpression of miR-222-3p and suppressors of cytokine signaling 1 (SOCS1) was detected using real-time quantitative PCR and western blot. Liver injury was determined by levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory cytokines, numbers of infiltrating mononuclear cells using AST/ALT assay kit, enzyme-linked immunosorbent assay (ELISA), and hematoxylin-eosin staining, respectively. Target binding between miR-222-3p and SOCS1 was predicted on targetScan software, and confirmed by luciferase reporter assay.ResultsSEB induced liver injury in D-galactosamine (D-gal)-sensitized mice, as demonstrated by increased serum levels of AST and ALT, elevated release of interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2, and promoted infiltrating immune cells into liver. Expression of miR-222-3p was dramatically upregulated, and SOCS1 was downregulated in SEB-induced liver injury both in mice and splenocytes. Moreover, miR-222-3p knockout (KO) mice exhibited alleviated liver injury accompanied with SOCS1 upregulation. Besides, splenocytes under SEB challenge released less INF-γ, TNF-α, IL-6, and IL-2 during miR-222-3p knockdown. Mechanically, SOCS1 was targeted and downregulated by miR-222-3p. Upregulation of SOCS1 attenuated INF-γ, TNF-α, IL-6, and IL-2 release in SEB-induced splenocytes; downregulation of SOCS1 could block the suppressive role of miR-222-3p knockdown in SEB-induced splenocytes.ConclusionInhibition of miR-222-3p relieves SEB-induced liver inflammatory injury by upregulating SOCS1, thereby providing the first evidence of miR-222-3p in SEB-induced liver injury.

A novel staphylococcal enterotoxin B subunit vaccine candidate elicits protective immune response in a mouse model

Staphylococcal enterotoxin B (SEB), produced by the gram-positive bacterium Staphylococcus aureus, is responsible for food poisoning and toxic shock syndrome, and is considered a potential bioterrorism agent. Unfortunately, still now no approved vaccines are available against SEB. In this study, we constructed a series of nontoxic SEB mutants (mSEBs) and examined whether these mSEBs provide protective immunity against SEB challenge. These mSEB vaccine candidates did not demonstrate superantigen activity in mouse splenocyte cultures. Immunization with the vaccine candidates triggered the production of IgG-antibodies with neutralizing activity. In addition, increased production of IgG1 and IgG3 was observed after immunization, which signifies both Th1- and Th2-induced immune responses. Among the vaccine candidates tested, S9, a double mutant (N23A and Y90A) and S19, a quadruple mutant (N23A, Y90A, R110A, and F177A), demonstrated complete protection against a lethal SEB challenge. Altogether, our results strongly suggest that these mSEBs could be an effective recombinant SEB vaccine candidates for further/future preclinical and clinical studies.

Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.

IL-33 and ST2 in Atopic Dermatitis: Expression Profiles and Modulation by Triggering Factors

In the acute phase of atopic dermatitis (AD), T-helper type 2 (Th2) cytokines characterize the inflammatory response in the skin. IL-33 is a new tissue-derived cytokine, which is mainly expressed by cells of barrier tissues, and is known to activate Th2 lymphocytes, mast cells, and eosinophils. IL-33 signals through a receptor complex consisting of IL-33-specific receptor ST2 and a co-receptor IL-1RAcP. As IL-33 is known to promote Th2-type immunity, we examined expression profiles of IL-33 and its receptor components in human AD skin, in the murine model of AD, and in various cell models. We found increased expression of IL-33 and ST2 in AD skin after allergen or staphylococcal enterotoxin B (SEB) exposure, as well as in the skin of 22-week-old filaggrin-deficient mice. In addition, skin fibroblasts, HaCaT keratinocytes, primary macrophages, and HUVEC endothelial cells efficiently produced IL-33 in response to the combined stimulation of tumor necrosis factor-α and IFN-γ, which was further enhanced by a mimetic of double-stranded RNA. Finally, the increased expression of IL-33 and ST2 caused by irritant, allergen, or SEB challenge was suppressed by topical tacrolimus treatment. These results suggest an important role for IL-33-ST2 interaction in AD and highlight the fact that bacterial and viral infections may increase the production of IL-33.

Generation and characterization of high affinity human monoclonal antibodies that neutralize staphylococcal enterotoxin B

BackgroundStaphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies.MethodsTo develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo.ResultsThe high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD50 (0.2 μg) , protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge.ConclusionWe have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.

Detrimental Effect of the Proteasome Inhibitor, Bortezomib in Bacterial Superantigen- and Lipopolysaccharide-induced Systemic Inflammation

Bacterial superantigen (BSAg)-induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)-induced shock are characterized by severe systemic inflammation. As nuclear factor kappaB (NF kappaB) plays an important role in inflammation and bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NF kappaB activation, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using murine models. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, at 3 hours, serum level of TNF-a, an important cytokine implicated in TSS, was significantly reduced but not abolished. At 6 hours, there was no difference in the serum TNF-a levels between bortezomib treated and untreated mice challenged with staphylococcal enterotoxin B (SEB). Paradoxically, all mice treated with bortezomib either before or after BSAg challenge succumbed to TSS. Neither bortezomib nor BSAg was lethal if given alone. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB-challenged mice treated with bortezomib showed a significant reduction in NF kappaB activation. Because NF kappaB-dependent antiapoptotic pathways protect hepatocytes from TNF-alpha-induced cell death, inhibition of NF kappaB brought forth by bortezomib in the face of elevated TNF-alpha levels caused by BSAg or LPS is detrimental.

Rapamycin Protects Mice from Staphylococcal Enterotoxin B-Induced Toxic Shock and Blocks Cytokine ReleaseInVitroandIn Vivo

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-2, gamma interferon (IFN-gamma), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.

Desensitized Renal Transplant Recipients Show Reduced Cellular Responses to In Vitro Challenge

Intravenous immunoglobulin (IVIG) desensitization protocols permit transplantation of sensitized renal recipients against alloantibody incompatibility barriers. It appears that IVIG induces a sustained down-regulation of alloantibody that facilitates crossmatch compatibility. However, the consequence of desensitization upon cellular immunity has not been addressed. We compared in vitro proliferative responses and cytokine expression between desensitized transplant recipients (IVIG) and non-IVIG-treated patients (controls). Eleven patients who were crossmatch-incompatible with living donors underwent treatment with IVIG and plasmapheresis. Nine patients converted to negative crossmatches and were transplanted. With a mean follow-up time of 17.6 ± 6.7 months, graft survival is 100%. Peripheral mononuclear cells were collected 12.5 ± 9 months after transplantation and tested for proliferative response and cytokine elaboration following challenge with staphylococcal enterotoxin b (SEB) and donor-specific mixed lymphocyte reaction (dMLR). Proliferative reactions were 90% lower ( P < .05) among IVIG versus controls in response to SEB and dMLR challenge. Cytokine response to SEB challenge was equivalent ( P = NS) between IVIG and controls. In contrast, cytokine elaboration was 60% lower ( P < .05) for IVIG versus controls in response to dMLR. One year after renal transplantation, desensitized patients showed in vitro hyporesponsiveness to superantigen and donor-specific challenge. This suggests that IVIG promotes a down-regulation in cellular immunity that may facilitate good graft outcome.

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.

Staphylococcal enterotoxin B-induced acute inflammation is inhibited by dexamethasone: important role of CXC chemokines KC and macrophage inflammatory protein 2.

This study was conducted to examine the anti-inflammatory mechanisms of dexamethasone during leukocyte recruitment and expression of the CXC chemokines macrophage inflammatory protein 2 (MIP-2) (CXCL2) and cytokine-induced neutrophil chemoattractant (KC) (CXCL1) in staphylococcal enterotoxin B (SEB)-induced acute inflammation. To do this, SEB was injected into murine air pouches with or without dexamethasone pretreatment for 2 h. SEB induced infiltration of leukocytes in a dose- and time-dependent manner, with the maximal response observed after 4 h of treatment with 10 microg of SEB. The recruited leukocytes comprised more than 77% neutrophils. Moreover, SEB challenge (10 microg) provoked time-dependent secretion of CXC chemokines, which peaked after 1 h. Local administration of antibodies against MIP-2 and KC significantly reduced SEB-triggered neutrophil accumulation by 38 and 59%, respectively. Dexamethasone (10 mg kg(-1)) significantly decreased neutrophil recruitment by 82% and reduced secretion of MIP-2 and KC by 89 and 85%, respectively, in response to SEB challenge. Our data demonstrate that dexamethasone potently inhibits neutrophil recruitment in SEB-induced inflammation. Moreover, we provide evidence that MIP-2 and KC are key mediators in the neutrophil response to SEB. Furthermore, our findings demonstrate that dexamethasone attenuates SEB-induced expression of MIP-2 and KC. Thus, this study elucidates important signaling pathways of SEB-induced neutrophil recruitment and anti-inflammatory mechanisms of action of dexamethasone.

Cellular and cytokine responses in the circulation and tissue reactions in the lung of rhesus monkeys (Macaca mulatta) pretreated with cyclosporin A and challenged with staphylococcal enterotoxin B.

Cyclosporin A (CsA), an inhibitor of T cell cytokine production, protects mice against staphylococcal enterotoxin B (SEB) intoxication. To determine whether CsA treatment would work in a species closer to humans. 4 rhesus monkeys were given 50 mg/kg CsA followed by an intratracheal challenge with approximately 6 LD50 of SEB. The CsA was not protective: one of the monkeys died and the other three had to be euthanised when they became moribund. All monkeys made IL-2, TNF, and IFN-gamma in response to SEB. In addition, there was about a 10-fold increase in ACTH levels 2 hr after SEB challenge. CsA significantly suppressed in vitro proliferation of lymphocytes from treated monkeys. Both CsA-treated monkeys and monkeys that had been challenged in a previous experiment with a lethal dose of SEB but had received no cyclosporin had pathologic changes in several organs. The most prominent changes were marked edema and leukocytic infiltration of the bronchial and bronchiolar mucosa. The CsA treatment appeared to reduce the intensity of lung inflammation, but this effect was not sufficient to protect the monkeys. The results suggest that CsA alone may not be an effective therapeutic agent for humans suffering from SEB intoxication or gram-positive septic shock.

A central role for CD95 (Fas) in T-cell reactivity after injury

Background: Recent findings indicate that severe injury primes the immune system for an enhanced and lethal proinflammatory cytokine response against bacterial-derived superantigens. This study asked whether this response to injury involves the CD95 (Fas) signaling pathway. Methods: To assess superantigen-mediated mortality, wild-type (WT) C57BL/6 and Fas-deficient C57BL/6 lpr (-/-) (lpr) mice underwent burn or sham injury and were challenged 2 hours later with staphylococcal enterotoxin B (SEB). Spleen cells from sham and burn WT or lpr mice were stimulated in vitro with SEB to assess injury effects on IL-2, TNF-α, and IFN-γ production. Results: Lpr burn mice survived the SEB challenge (100% survival), while WT burn mice showed a high mortality (17% survival, P < 001, analysis of variance [ANOVA]). Sham lpr or WT mice suffered no mortality to the SEB challenge. In vitro studies demonstrated that burn lpr mice produced significantly less TNF-α, IFN-γ, IL-2 than burn WT mice (P <.01, ANOVA). Burn injury markedly enhanced SEB-stimulated IFN-γ production by WT spleen cells and CD8+ T cells, while this did not occur in SEB-stimulated lpr spleen cells. Conclusions: These findings support the hypothesis that the CD95 (Fas) signaling pathway plays an integral role in the injury-induced enhanced and lethal T-cell reactivity against bacterial superantigens. (Surgery 2000;128:159-64.)

T-Lymphocyte Activation Increases Hypothalamic and Amygdaloid Expression of CRH mRNA and Emotional Reactivity to Novelty

Stimulation of T-cells with staphylococcal enterotoxin B (SEB) significantly elevates interleukin-2 (IL-2) and contemporaneous activation of the hypothalamic-pituitary-adrenal (HPA) axis and c-fos in the paraventricular nucleus (PVN) of BALB/cByJ mice. Such neural signaling may promote cognitive and emotional adaptation before or during infectious illness. Because corticotropin-releasing hormone (CRH) is an anxiogenic neuropeptide that may mediate the stressor-like effects of immunological stimuli, we measured neuronal CRH mRNA alterations in mice challenged with SEB. Increased CRH mRNA levels were observed in the PVN and central nucleus of the amygdala (ceA) 4-6 hr after SEB administration. This was associated with plasma ACTH increases, which could be abrogated by the systemic administration of anti-CRH antiserum. Additional experiments did not support a role for IL-2 or prostaglandin synthesis in activating the HPA axis. Behavioral experiments testing for conditioned taste aversion did not confirm that SEB challenge promotes malaise. However, consistent with the notion that central CRH alterations induced by SEB may affect emotionality (e.g., fear), SEB challenge augmented appetitive neophobia in a context-dependent manner, being marked in a novel and stressful environment. It is hypothesized that immunological stimuli generate a cascade of events that solicit integrative neural processes involved in emotional behavior. As such, these data support the contention that affective illness may be influenced by immunological processes and the production of cytokines and are consistent with other evidence demonstrating that autoimmune reactivity is associated with enhanced emotionality.

In Vivo Effects of a Bacterial Superantigen on Macaque TCR Repertoires

A macaque model was employed to explore staphylococcal enterotoxin B (SEB) superantigen-driven T lymphocyte responses. The SEB-reactive Vbeta+ cell subpopulations demonstrated a striking tri-phase response in rhesus monkeys following an SEB challenge in vivo. The hyperacute down-regulation, seen as early as 2 h through 2 days after SEB injection, was characterized by a disappearance of the reactive Vbeta-restricted PBL subpopulations from the circulation and decreased expression of these cell subpopulations in lymphoid tissues. Following this, a dominant expansion of reactive Vbeta-expressing CD4+ cell subpopulations occurred in lymph nodes and spleens, whereas in the peripheral blood a preferential expansion of reactive Vbeta-expressing CD8+ cell subpopulations was seen. An exhaustion of this response was then seen, with a prolonged decrease in the number of the reactive Vbeta+ CD4+ lymphocyte subpopulations. Interestingly, monoclonal or oligoclonal dominance was seen in the reactive Vbeta+ cell subpopulations in the period of the transition from the polyclonal cellular expansion to the exhaustion of the response, suggesting that some Vbeta+ cell clones may be more resistant than others to superantigen-mediated depletion. These results indicate that in vivo SEB superantigen-mediated effect on lymphocyte subpopulations in macaques is complex, suggesting that profound dynamics in the TCR repertoires may in part account for the susceptibility of higher primates to SEB-induced diseases.

The crucial role of IL-10 in the suppression of the immunological response in mice exposed to staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB), a bacterial superantigen, activates the immune system resulting in a burst of pro- and anti-inflammatory cytokines. A central anti-inflammatory mediator in this process is IL-10. Using IL-10-deficient C57BL/6 (IL-10 KO) mice, we studied the role of endogenous IL-10 in the regulation of the immune response to SEB. SEB (100 microg) induced the release of IL-10 in control C57BL/6 [IL-10 wild type (WT)] mice, but not in their IL-10 KO counterparts. SEB-evoked plasma levels of TNF-alpha, IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma were significantly higher in the IL-10 KO mice than in the WT animals. The release of macrophage inflammatory proteins-1alpha and -2 was also enhanced in the IL-10 KO mice. Further, upon SEB challenge, mice deficient in IL-10 produced higher levels of nitric oxide than the WT animals. IL-10 deficiency resulted in a marked enhancement of the SEB-induced apoptosis of thymocytes. Finally, IL-10 KO mice were more susceptible to SEB-induced lethal shock than their WT controls. These results show that IL-10 plays an important immunoregulatory role in the response to a superantigenic stimulus, by dampening of the shock-inducing inflammatory response and early activation-induced cell death elicited by SEB.

Differential immune responses to staphylococcal enterotoxin B mutations in a hydrophobic loop dominating the interface with major histocompatibility complex class II receptors.

Bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can trigger acute pathologic effects in humans. A hydrophobic loop on the surface of SEB and other bacterial superantigens, centered around a conserved leucine (L45) residue, is essential for binding to class II major histocompatibility complex molecules. Single residue changes of wild type SEB, designated Q43P, F44P, or L45R, resulted in nonlethal proteins at a dose equivalent to 30 murine LD50 of SEB. Relative to SEB, the mutant proteins did not elevate serum concentrations of proinflammatory cytokines in mice and caused minimal proliferation of human lymphocytes. Anti-SEB titers of mice immunized with Q43P, F44P, L45R, or SEB were similar and protected 77%-100% of animals against a lethal SEB challenge. Levels of toxin-specific IgG1, IgG2a, IgG2b, and IgG3 in mice immunized with SEB, Q43P, or F44P were equivalent, but animals immunized with L45R had significantly elevated levels of IgG2a and IgG2b. Vaccines against staphylococcal superantigens should focus on this critical leucine residue.

Immediate responses of leukocytes, cytokines and glucocorticoid hormones in the blood circulation of monkeys following challenge with aerosolized staphylococcal enterotoxin B.

The immediate responses to aerosolized staphylococcal enterotoxin B (SEB) in respiratory toxic shock were studied in the circulation of rhesus monkeys with low antibody levels following immunization with SEB toxoid-containing microspheres. Both the surviving and dying monkeys had toxic shock syndrome 4-48 h after SEB challenge and all showed three distinctive patterns of immediate responses. The first pattern, characterized by the responses of all T cells, HLA-DRlo cells, monocytes, IL-2R+ cells, IFN-gamma, and augmented lymphocyte mitotic responses to lipopolysaccharide (LPS) and SEB in culture, was a rapid increase at 20 min followed by a quick decrease at 90 min to approximately the original levels. The second pattern, which included responses of HLA-DRhi cells, NK cells, adrenocorticotropic hormone (ACTH) and cortisol, was characterized by a moderate decrease at 20 min and a further decrease at 90 min. The third pattern, the inverse of the second pattern, including responses of polymorphonuclear leukocytes (PMN), concanavalin A (Con A) mitogenesis, IL-6 and IL-2, was a moderate increase at 20 min and a further increase at 90 min. Between the surviving and dying monkeys, the responses of T cells, HLA-DRhi cells, PMN and cortisol did not differ significantly, suggesting that they are the basic causes that initiated toxic shock. However, significant differences were seen in the responses of HLA-DRlo cells, monocytes, IL-2R+ cells and lymphocyte mitogenesis in culture at 20 min, and of Con A mitogenesis, NK cells, IL-2, IL-6 and ACTH at 90 min. These different responses are apparently the exacerbating causes of death of the monkeys. All together, the immediate responses seem to be caused by the combined effects of SEB superantigenicity, activation of NK cells and non-lymphoid cells, and depression of the neuroimmune defense system.