High Titer Persistent Neutralizing Antibodies Induced by TSST-1 Variant Vaccine Against Toxic Shock Cytokine Storm.

Staphylococcal superantigen toxins lead to a devastating cytokine storm resulting in shock and multi-organ failure. We have previously assessed the safety and immunogenicity of a recombinant toxic shock syndrome toxin 1 variant vaccine (rTSST-1v) in clinical trials (NCT02971670 and NCT02340338). The current study assessed neutralizing antibody titers after repeated vaccination with escalating doses of rTSST-1v. At study entry, 23 out of 34 subjects (67.6%) had neutralizing antibody titers inhibiting T cell activation as determined by 3H-thymidine incorporation at a serum dilution of ≤1:100 with similar figures for inhibition of IL-2 activation (19 of 34 subjects, 55.9%) as assessed by quantitative PCR. After the first vaccination, numbers of subjects with neutralization titers inhibiting T cell activation (61.7% ≥ 1:1000) and inhibiting IL-2 gene induction (88.2% ≥ 1:1000) increased. The immune response was augmented after the second vaccination (inhibiting T cell activation: 78.8% ≥ 1:1000; inhibiting IL-2 induction: 93.9% ≥ 1:1000) corroborated with a third immunization months later in a small subgroup of subjects. Assessment of IFNγ, TNFα and IL-6 inhibition revealed similar results, whereas neutralization titers did not change in placebo participants. Antibody titer studies show that vaccination with rTSST-1v in subjects with no/low neutralizing antibodies can rapidly induce high titer neutralizing antibodies persisting over months.

An iRGD peptide fused superantigen mutant induced tumor-targeting and T lymphocyte infiltrating in cancer immunotherapy.

Solid tumors are intrinsically resistant to immunotherapy because of the major challenges including the immunosuppression and poor penetration of drugs and lymphocytes into solid tumors due to the complicated tumor microenvironment (TME). Our previous study has created a novel superantigen mutant ST-4 to efficiently active the T lymphocytes and alleviate immune suppression. In the present study, to accumulate ST-4 into the TME, we constructed a recombinant protein, ST-4-iRGD, by fusing ST-4 to a tumor-homing peptide, iRGD. We hypothesized that ST-4-iRGD could internalize into the TME through iRGD-mediated tumor targeting and tumor tissue penetrating to activate the regional immunoreaction. The results of in vitro studies showed that ST-4-iRGD achieved improved tumor targeting and cytotoxicity in mouse B16F10 melanoma cells. The iRGD-mediated tumor tissue penetration was further confirmed by imaging and immunofluorescence studies in vivo, wherein higher distribution of ST-4-iRGD was observed in the mouse 4T1 breast tumor model. Moreover, ST-4-iRGD exhibited enhanced anti-solid tumor characteristics and induced improved lymphocyte infiltration in the B16F10 and 4T1 models. In conclusion, using iRGD to facilitate better dissemination of the therapeutic agent ST-4 throughout a solid tumor mass is feasible, and ST-4-iRGD may be a potential candidate for efficient cancer immunotherapy in the future.

Interleukin-10 (IL-10) Produced by Mutant Toxic Shock Syndrome Toxin 1 Vaccine-Induced Memory T Cells Downregulates IL-17 Production and Abrogates the Protective Effect against Staphylococcus aureus Infection.

Development of long-term memory is crucial for vaccine-induced adaptive immunity against infectious diseases such as Staphylococcus aureus infection. Toxic shock syndrome toxin 1 (TSST-1), one of the superantigens produced by S. aureus, is a possible vaccine candidate against infectious diseases caused by this pathogen. We previously reported that vaccination with less toxic mutant TSST-1 (mTSST-1) induced T helper 17 (Th17) cells and elicited interleukin-17A (IL-17A)-mediated protection against S. aureus infection 1 week after vaccination. In the present study, we investigated the host immune response induced by mTSST-1 vaccination in the memory phase, 12 weeks after the final vaccination. The protective effect and IL-17A production after vaccination with mTSST-1 were eliminated because of IL-10 production. In the presence of IL-10-neutralizing monoclonal antibody (mAb), IL-17A production was restored in culture supernatants of CD4+ T cells and macrophages sorted from the spleens of vaccinated mice. Vaccinated mice treated with anti-IL-10 mAb were protected against systemic S. aureus infection in the memory phase. From these results, it was suggested that IL-10 produced in the memory phase suppresses the IL-17A-dependent vaccine effect through downregulation of IL-17A production.

Macrophage migration inhibitory factor regulates innate γδ T-cell responses via IL-17 expression.

T cells expressing invariant γδ antigen receptors (γδ T cells) bridge innate and adaptive immunity and facilitate barrier responses to pathogens. Macrophage migration inhibitory factor (MIF) is an upstream mediator of host defense that up-regulates the expression of pattern recognition receptors and sustains inflammatory responses by inhibiting activation-induced apoptosis in monocytes and macrophages. Surprisingly, Mif-/- γδ T cells, when compared with wild type, were observed to produce >10-fold higher levels of the proinflammatory cytokine IL-17 after stimulation with gram-positive exotoxins. High-IL-17 expression was associated with the characteristic features of IL-17-producing γδ T (γδ17) cells, including expression of IL-23R, IL-1R1, and the transcription factors RORγt and Sox13. In the gram-positive model of shock mediated by toxic shock syndrome toxin (TSST-1), Mif-/- mice succumbed to death more quickly with increased pulmonary neutrophil accumulation and higher production of cytokines, including IL-1ß and IL-23. Mif-/- γδ T cells also produced high levels of IL-17 in response to Mycobacterium lipomannan, and depletion of γδ T cells improved survival from acutely lethal Mycobacterium infection or TSST-1 administration. These data indicate that MIF deficiency is associated with a compensatory amplification of γδ17 cell responses, with implications for innate immunity and IL-17-mediated pathology in situations such as gram-positive toxic shock or Mycobacterium infection.-Kim, H. K., Garcia, A. B., Siu, E., Tilstam, P., Das, R., Roberts, S., Leng, L., Bucala, R. Macrophage migration inhibitory factor regulates innate γδ T-cell responses via IL-17 expression.

Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer therapy.

Recent research has attempted to direct superantigens towards tumors by means of tumor-targeted superantigen (TTS) strategy. In this study, we explored the antitumor property of TTS by fusing the third loop of transforming growth factor α (TGFαL3) to staphylococcal enterotoxin type B (SEB) and investigated the possibility of the therapeutic application of TGFαL3-SEB as a novel antitumor candidate in mice bearing breast cancer. Treatment was performed through intratumoral and intravenous injection of TGFαL3-SEB. Tumor size/volume, long-term survival, and cytokine secretion were assessed. In addition, the toxicity of each treatment on liver and kidneys was examined. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral TGFaL3-SEB (p < 0.05). Surprisingly, 5 out of the 14 mice were cleared from the tumor thoroughly in 10-25 days after intratumoral administration of TGFaL3-SEB. Quantification of cytokines clearly showed that the mice receiving intratumoral SEB significantly secreted higher interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the other groups (p < 0.05). The antitumor effect was followed by inhibition of cell proliferation (Ki-67) and micro vascularization (CD31). The highest and lowest levels of tumor necrosis were observed in the intratumoral administration of TGFαL3-SEB (85 %) and PBS (14 %), respectively. Intratumoral injection of TGFαL3-SEB increased the lifespan of the mice so 37.5 % of them could survive for more than 6 months (p < 0.05). Overall, our findings indicated that intratumoral administration of TGFαL3-SEB effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis without systemic toxicity.

Mini-Osmotic Pump Infusion Model to Investigate the Systemic Effects of Chronic Continuous Exposure to Staphylococcal Superantigen in Mice.

Staphylococcus aureus can exist as a colonizer or can cause a spectrum of diseases. S. aureus elaborates several exotoxins and the superantigens are one among them. Staphylococcal superantigens (SSAg) cause robust activation of the immune system and acute exposure to significant amounts of SSAg can be potentially lethal. However, chronic exposure to SSAg is also possible. Administering SSAg using mini-osmotic pumps may mimic chronic recurrent exposure to SSAg. This is a relatively simple and safe way to administer purified SSAg or any other toxin/agent. In this chapter, we describe the mini-osmotic pump-mediated delivery of SSAg.

Analysis of the treatment of gliomas with SEC therapy combined with radiochemotherapy.

OBJECTIVE: To investigate the anti-glioma effects of staphylococcal enterotoxin C (SEC) combined with surgery, radiotherapy and chemotherapy, and the IL-2 of lymphocytes activated by SEC. PATIENTS AND METHODS: A total of 92 patients with gliomas which had been surgically removed, were randomly divided into a control, and treatment groups A (systemic application of SEC) and B (local application of lymphocytes activated by SEC). The treatment effects were observed on a CT scan of the head. The SEC's induction on the IL-2 secretion by lymphocytes was determined using immunohistochemistry and RT-PCR methods. RESULTS: Clinical materials showed that the effective rate (CR and PR) in the control group was 32.0%. In the treatment group A, the effective rate was 51.6% and 63.6% in the group B. The IL-2 of lymphocytes secreting activity activated by SEC began to secrete IL-2 on the 1st day and reached a peak during the 3rd day and the 5th day. CONCLUSIONS: As a superantigen, SEC combined with surgery, radiotherapy and chemotherapy can improve treatment. The lymphocytes activated by SEC can release IL-2.

Chronic exposure to staphylococcal superantigen elicits a systemic inflammatory disease mimicking lupus.

Chronic nasal and skin colonization with superantigen (SAg)-producing Staphylococcus aureus is well documented in humans. Given that trans-mucosal and trans-cutaneous absorption of SAgs can occur, we determined whether chronic exposure to small amounts of SAg per se could activate autoreactive CD4(+) and CD8(+) T cells and precipitate any autoimmune disease without further external autoantigenic stimulation. Because HLA class II molecules present SAg more efficiently than do mouse MHC class II molecules, HLA-DQ8 transgenic mice were implanted s.c. with mini-osmotic pumps capable of continuously delivering the SAg, staphylococcal enterotoxin B (total of 10 µg/mouse), or PBS over 4 wk. Chronic exposure to staphylococcal enterotoxin B resulted in a multisystem autoimmune inflammatory disease with features similar to systemic lupus erythematosus. The disease was characterized by mononuclear cell infiltration of lungs, liver, and kidneys, accompanied by the production of anti-nuclear Abs and deposition of immune complexes in the renal glomeruli. The inflammatory infiltrates in various organs predominantly consisted of CD4(+) T cells bearing TCR Vß8. The extent of immunopathology was markedly reduced in mice lacking CD4(+) T cells and CD28, indicating that the disease is CD4(+) T cell mediated and CD28 dependent. The absence of disease in STAT4-deficient, as well as IFN-γ-deficient, HLA-DQ8 mice suggested the pathogenic role of Th1-type cytokines, IL-12 and IFN-γ. In conclusion, our study suggests that chronic exposure to extremely small amounts of bacterial SAg could be an etiological factor for systemic lupus erythematosus.

Immunity to Staphylococcus aureus secreted proteins protects rabbits from serious illnesses.

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. This study investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50% of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (ß-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins ß-toxin and γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.

Specific immunosuppression by mixed chimerism with bone marrow transplantation after Staphylococcal Enterotoxin B pretreatment could prolong corneal allograft survival in mice.

PURPOSE: We assessed the combined use of Staphylococcal Enterotoxin B (SEB) superantigen pre-treatment along with allogeneic bone marrow transplant (BMT) to induce immune suppression condition and inhibit corneal keratoplasty rejection in mice. METHODS: BALB/C (H-2d) mice were both BMT and corneal allografts donors and C57BL/6(H-2b) mice were recipients. Prior to BMT, recipients received single injections of either SEB, cyclophosphamide (CYP), or normal saline (NS). Allogenic corneal penetrating keratoplasty was performed 7 days after BMT. Bone marrow chimerisms in recipients (donor major histocompatibility complex-II H2-d) were determined on Days 14, 28, and 56 post-BMT. Recipient immune response was assessed by mixed lymphocyte reactions (MLR) using splenocytes from C57BL/6 mice as responders in co-culture with stimulator cells from C57BL/6 (isogeneic), BALB/C (allogeneic), or CBA/1(third party) mice. Cluster of differentiation 4 receptors positive (CD4+) and CD8+T cells in recipient mice were evaluated. Corneal graft survival was assessed using Kaplan-Meier survival curves. RESULTS: SEB pre-treatment induced higher levels of hematopoietic chimerism on Days 14, 28 and 56 post-BMT than did CYP or NS pre-treatment. Mean corneal allograft survival was significantly prolonged with group SEB-BMT (20.3±7.6 days) compared to group CYP-BMT (13.0±4.0 days) and NS-BMT (9.0±2.2 days). SEB-BMT mice splenocytes had diminished MLR responses compared to CYP-BMT or NS-BMT mice. CD4+ and CD8+ T cells in peripheral blood and spleens were significantly reduced in group SEB-BMT mice. CONCLUSIONS: BMT after SEB pre-treatment could promote mixed chimerism, which inhibited allogeneic cornea transplant rejection. This should possibly relate to CD4+ and CD8+ T cell deletion and acquiring donor-specific immunosuppression.

Staphylococcal enterotoxin A has potent superantigenic and emetic activities but not diarrheagenic activity.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are pyrogenic superantigenic toxins that are involved in human diseases including food poisoning and toxic shock syndrome. Although the superantigenic activity of SEs has been well characterized, its role and mechanism in clinical symptoms of food poisoning remain poorly understood. In this study, house musk shrews (Suncus murinus), a small emetic animal model, were used to study the role of SEs in clinical manifestations of food poisoning. Administration of SEA induced a potent emetic response in vivo and showed significant superantigenic activity in vitro in house musk shrews. However, SEA revealed no diarrheagenic activity. SEA directly injected into the intestinal loops of house musk shrews failed to induce fluid exudation and consequent dilation of the intestinal segments. Rabbit intestinal loop experiments were further carried out to confirm the results and also showed that SEA induced no fluid exudation and consequent dilation. Furthermore, the SEA-producing S. aureus also failed to induce fluid exudation in the administered loops of these animal models. These results indicate that SEA has potent superantigenic and emetic activities, but does not have a diarrheagenic activity.

Bacterial superantigens enhance the in vitro proinflammatory response and in vivo lethality of the TLR2 agonist bacterial lipoprotein.

Bacterial superantigens are Gram-positive exotoxins that induce proinflammatory cytokine release in vitro, cause lethal shock in vivo, and can be detected in the bloodstream of critically ill patients. They also have a powerful priming effect on the TLR4 agonist LPS. The aim of this study was to investigate the relationship between superantigens and the TLR2 agonist bacterial lipoprotein (BLP). Priming of human monocytes or PBMCs with superantigens significantly enhanced proinflammatory cytokine TNF-α and IL-6 release in response to BLP stimulation. The priming effect of superantigens could be blocked by inhibiting p38 MAPK during the priming phase as opposed to NF-κB or ERK inhibition. This was consistent with higher expression of the phosphorylated p38 after superantigen priming and BLP or LPS stimulation. C57BL/6 mice with superantigen priming (10 µg/mouse) when challenged with BLP (600 µg/mouse) exhibited substantially higher mortality (100%) compared with mice without superantigen priming (zero). Mice given superantigen alone did not demonstrate any signs of illness. Mice challenged with both superantigen and BLP had significantly higher levels of serum TNF-α and IL-6 compared with those of mice challenged with either agent alone. Depletion of the monocyte/macrophage subpopulation significantly reduced the mortality rate from 100 to 20% in superantigen-primed, BLP-challenged C57BL/6 mice, with a 5- to 10-fold decrease in serum TNF-α and IL-6. Our results demonstrate that bacterial superantigens enhance the in vitro proinflammatory cytokine release and in vivo lethality of BLP. This novel finding may help to explain the massive proinflammatory cytokine release seen in superantigen-mediated septic shock.

Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock.

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved ß-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the ß-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.

DNAM-1 ligand expression on Ag-stimulated T lymphocytes is mediated by ROS-dependent activation of DNA-damage response: relevance for NK-T cell interaction.

An important role for natural killer (NK) cells in the regulation of T-cell responses is emerging, although the receptor pairs regulating the NK-T-cell interaction have still not been identified. We found that superantigen-stimulated T cells express Nectin-2 (CD112) and poliovirus receptor (PVR; CD155), the ligands of the activating NK receptor DNAX accessory molecule-1 (DNAM-1; CD226). Interestingly, only PVR was present at the T cell surface, particularly on cells in the S and G(2)/M phases of the cell cycle. The up-regulation of PVR expression involves DNA-damage response (DDR)-dependent pathways, because we found that pharmacologic inhibition of ATM and ATR kinases reduced PVR expression and that PVR was almost exclusively induced on cells expressing the DDR marker γH2AX. Oxidative stress contributed to DDR activation, and our results showed impaired PVR levels in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), being monocytes the main ROS source needed for optimal PVR expression on activated T cells. Interestingly, in accordance with ligand expression, NK cells lysed allogeneic proliferating more efficiently than nonproliferating T lymphocytes, with a mechanism requiring the cooperation between DNAM-1 and NKG2D. These results could contribute to unraveling the role of NK cells in the down-regulation of T-cell responses in physiologic and pathologic processes such as autoimmunity or GVHD.

Murine models of staphylococcal enterotoxin B-induced toxic shock.

Staphylococcal enterotoxin B (SEB) is a member of a large family of structurally related exotoxins produced by Staphylococcus aureus, which is the etiological agent responsible for toxic shock and staphylococcal food poisoning. SEB binds directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and T-cell receptors on T cells triggering T-cell proliferation and mediator release. SEB is a biothreat agent because of its ability to potently activate cells of the immune system. In vivo animal models are critical in the development of therapeutics against SEB-induced shock. Our results show that three different mouse strains with different susceptibility to SEB can be used to study SEB-induced shock without the use of potentiating agents. The hypothermic response, weight loss, and induction of serum monocyte chemoattractant protein 1 (MCP-1), interleukin 2 (IL-2), and IL-6 correlated with mortality in all three models.

Staphylococcal superantigens cause lethal pulmonary disease in rabbits.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) and others reported that methicillin-resistant S. aureus (MRSA) are significant causes of serious human infections, including pulmonary illnesses. We investigated the role played by superantigens in lung-associated lethal illness in rabbits. METHODS: A rabbit model was established to investigate the potential role played by superantigens, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), and toxic shock syndrome toxin-1 (TSST-1). Rabbits received intrabronchial community-associated (CA) MRSA strains USA200 (TSST-1(+)), MW2 (SEC(+)), c99-529 (SEB(+)), or purified superantigens. Some rabbits were preimmunized against superantigens or treated with soluble high-affinity T cell receptors (Vß-TCR) to neutralize SEB and then challenged intrabronchially with CA-MRSA or superantigens. RESULTS: Rabbits challenged with CA-MRSA or superantigens developed fatal, pulmonary illnesses. Animals preimmunized against purified superantigens, or treated passively with Vß-TCRs and then challenged with CA-MRSA or superantigens, survived. Lung histological analysis indicated that nonimmune animals developed lesions consistent with necrotizing pneumonia after challenge with CA-MRSA or purified superantigens. Superantigen-immune animals or animals treated with soluble Vß-TCRs did not develop pulmonary lesions. CONCLUSIONS: Superantigens contribute to lethal pulmonary illnesses due to CA-MRSA; preexisting immunity to superantigens prevents lethality. Administration of high-affinity Vß-TCR with specificity for SEB to nonimmune animals protects from lethal pulmonary illness resulting from SEB(+) CA-MRSA and SEB.

Oral vaccine formulations stimulate mucosal and systemic antibody responses against staphylococcal enterotoxin B in a piglet model.

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets 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 in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion 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.

T-cell subsets of the encephalitis induced by the superantigen Staphylococcal Enterotoxin A (SEA) in the Lewis rat: an immunohistochemical investigation.

In the present investigation, T-cell subsets of the previously described superantigen-induced encephalitis [9] have been investigated in 16 Lewis rats in comparison with four controls. Three days after intracerebral injection of Staphylococcal Enterotoxin A (SEA) or saline, 1.5 x 10(7) ConA-activated splenocytes were loaded i.v. animals were sacrificed after 0.5, 3 or 5 days, followed by immunohistochemical investigation of CD3, CD4 and CD8. Pronounced perivascular cuffing was identified 0.5 days after splenocyte injection and declined thereafter. The majority of the perivascular round cells consisted of CD8+ T-cells (65%) and CD4+ T-cells (10%). Less than 20% of the perivascular round cells were CD3+. The reduced expression of CD3 relative to e.g. CD8 is presumably due to the previous superantigenic stimulus. The presented data may be of relevance for the pathogenesis of infectious or autoimmune encephalitis, e.g. in multiple sclerosis.

Naptumomab estafenatox, an engineered antibody-superantigen fusion protein with low toxicity and reduced antigenicity.

Antibody-targeted superantigens have a potential to become useful drugs for tumor therapy. However, clinical practice has identified several issues that need to be addressed to optimize such molecules. On the basis of the experience from superantigen products in clinical trials, a novel tumor-targeted superantigen, naptumomab estafenatox (5T4FabV18-SEA/E-120 or ABR-217620) has been designed. Critical properties, such as tumor reactivity, therapeutic window, and seroreactivity were all improved. The engineered 5T4Fab moiety recognizes the 5T4 antigen expressed on a large number of solid tumor forms with an affinity in the order of 1 nM. The fusion protein induces T-cell mediated killing of tumor cells at concentrations around 10 pM. Compared with a construct with a wild-type superantigen, it is more potent in mediating killing of tumor cells but a 10,000-fold less active in mediating killing of MHC class II positive cells. The target epitopes for naturally occurring antibodies toward bacterial superantigens are reduced. Only large excesses of human anti-SEA antibodies neutralize the antitumor effects of the antibody-targeted superantigen. Naptumomab estafenatox induces dramatic reduction of established human tumors in Severe Combined Immunodeficient mice grafted with human lymphocytes. Thus, naptumomab estafenatox is a novel optimized tumor-targeted superantigen currently investigated in clinical trials.

Early gene expression changes induced by the bacterial superantigen staphylococcal enterotoxin B and its modulation by a proteasome inhibitor.

Toxic shock syndrome (TSS) is an acute, serious systemic illness caused by bacterial superantigens. Nonavailability of a suitable animal model until recently has hampered an in-depth understanding of the pathogenesis of TSS. In the current study, we characterized the early molecular events underlying TSS using our HLA-DR3 transgenic mouse model. Gene expression profiling using DNA microarrays identified a rapid and significant upregulation of several pro- as well as anti-inflammatory mediators, many of which have never been previously described in TSS. In vivo administration of staphylococcal enterotoxin B (SEB) led to an increase in the expression of Th0- (IL-2, 240-fold); Th1- (IFN-gamma, 360-fold; IL-12, 8-fold); Th2- (IL-4, 53-fold; IL-5, 4-fold) as well as Th17-type cytokines (IL-21, 19-fold; IL-17, 5-fold). The immunoregulatory cytokines (IL-6, 700-fold; IL-10, 18-fold); CC chemokines (such as CCL 2, 11, 3, 24, 17, 12, 7), CXC chemokines (such as CXCL 1, 2, 5, 11, 10, 19); and several proteases (matrix metalloproteinases 13, 8, 3, and 9) were also upregulated. Serum levels of several of these cytokines/chemokines were also significantly elevated. Pathway analyses revealed significant modulation in a variety of biochemical and cellular functions, providing molecular insights into the pathogenesis of TSS. Administration of bortezomib, a clinically approved proteasome inhibitor capable of blocking NF-kappaB pathway, was able to significantly modulate the expression of a variety of genes induced by SEB. Thus, our study showed that TSS is a complex process and emphasized the potential of use of bortezomib in the therapy of superantigen-induced TSS.