Superantigens promote Staphylococcus aureus bloodstream infection by eliciting pathogenic interferon-gamma production.

Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of exotoxins from S. aureus that bind directly to major histocompatibility complex (MHC) class II and T cell receptors to drive extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease, including toxic shock syndrome, the specific pathological mechanisms remain unclear. Herein, we aimed to elucidate how SAgs contribute to pathogenesis during bloodstream infections and utilized transgenic mice encoding human MHC class II to render mice susceptible to SAg activity. We demonstrate that SAgs contribute to S. aureus bacteremia by massively increasing bacterial burden in the liver, and this was mediated by CD4+ T cells that produced interferon gamma (IFN-γ) to high levels in a SAg-dependent manner. Bacterial burdens were reduced by blocking IFN-γ, phenocopying SAg-deletion mutant strains, and inhibiting a proinflammatory response. Infection kinetics and flow cytometry analyses suggested that this was a macrophage-driven mechanism, which was confirmed through macrophage-depletion experiments. Experiments in human cells demonstrated that excessive IFN-γ allowed S. aureus to replicate efficiently within macrophages. This indicates that SAgs promote bacterial survival by manipulating the immune response to inhibit effective clearing of S. aureus Altogether, this work implicates SAg toxins as critical therapeutic targets for preventing persistent or severe S. aureus disease.

Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens.

Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.

Impaired T-Lymphocyte Responses During Childhood Staphylococcus aureus Infection.

BACKGROUND: Staphylococcus aureus infections are common throughout the lifespan, with recurrent infections occurring in nearly half of infected children. There is no licensed vaccine, underscoring the need to better understand how S. aureus evades protective immunity. Despite much study, the relative contributions of antibodies and T cells to protection against S. aureus infections in humans are not fully understood. METHODS: We prospectively quantified S. aureus-specific antibody levels by ELISA and T-cell responses by ELISpot in S. aureus-infected and healthy children. RESULTS: S. aureus-specific antibody levels and T-cell responses increased with age in healthy children, suggesting a coordinated development of anti-staphylococcal immunity. Antibody levels against leukotoxin E (LukE) and Panton-Valentine leukocidin (LukS-PV), but not α-hemolysin (Hla), were higher in younger infected children, compared with healthy children; these differences disappeared in older children. We observed a striking impairment of global and S. aureus-specific T-cell function in children with invasive and noninvasive infection, suggesting that S. aureus-specific immune responses are dysregulated during childhood infection regardless of the infection phenotype. CONCLUSIONS: These findings identify a potential mechanism by which S. aureus infection actively evades adaptive immune responses, thereby preventing the development of protective immunity and maintaining susceptibility to recurrent infection.

Neutralization of the Staphylococcus aureus Panton-Valentine leukocidin by African and Caucasian sera.

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.

Depletion of regulatory T cells in tumors with an anti-CD25 immunotoxin induces CD8 T cell-mediated systemic antitumor immunity.

The tumor microenvironment plays a critical role in controlling tumor progression and immune surveillance. We produced an immunotoxin (2E4-PE38) that kills mouse cells expressing CD25 by attaching the Fv portion of monoclonal antibody 2E4 (anti-mouse CD25) to a 38-kDa portion of Pseudomonas exotoxin A. We employed three mouse cancer tumor models (AB1 mesothelioma, 66c14 breast cancer, and CT26M colon cancer). Tumors were implanted at two sites on BALB/c mice. On days 5 and 9, one tumor was directly injected with 2E4-PE38, and the other was not treated; 2E4-PE38 produced complete regressions of 85% of injected AB1 tumors, 100% of 66c14 tumors, and 100% of CT26M tumors. It also produced complete regressions of 77% of uninjected AB1 tumors, 47% of 66c14 tumors, and 92% of CT26M tumors. Mice with complete regressions of 66c14 tumors were immune to rechallenge with 66c14 cells. Mice with complete regressions of AB1 or CT26M tumors developed cross-tumor immunity rejecting both tumor types. Injection of anti-CD25 antibody or a mutant inactive immunotoxin were generally ineffective. Tumors were analyzed 3 days after 2E4-PE38 injection. The number of regulatory T cells (Tregs) was significantly reduced in the injected tumor but not in the spleen. Injected tumors contained an increase in CD8 T cells expressing IFN-γ, the activation markers CD69 and CD25, and macrophages and conventional dendritic cells. Treatment with antibodies to CD8 abolished the antitumor effect. Selective depletion of Tregs in tumors facilitates the development of a CD8 T cell-dependent antitumor effect in three mouse models.

Development of Combination Vaccine Conferring Optimal Protection against Six Pore-Forming Toxins of Staphylococcus aureus.

In the Gram-positive pathogen Staphylococcus aureus, pore-forming toxins (PFTs), such as leukocidins and hemolysins, play prominent roles in staphylococcal pathogenesis by killing host immune cells and red blood cells (RBCs). However, it remains unknown which combination of toxin antigens would induce the broadest protective immune response against those toxins. In this study, by targeting six major staphylococcal PFTs (i.e., gamma-hemolysin AB [HlgAB], gamma-hemolysin CB [HlgCB], leukocidin AB [LukAB], leukocidin ED [LukED], Panton-Valentine leukocidin [LukSF-PV], and alpha-hemolysin [Hla]), we generated 10 recombinant toxins or toxin subunits, 3 toxoids, and their rabbit antibodies. Using the cytolytic assay for RBCs and polymorphonuclear cells (PMNs), we determined the best combination of toxin antibodies conferring the broadest protection against those staphylococcal PFTs. Although anti-HlgA IgG (HlgA-IgG) showed low cross-reactivity to other toxin components, it was essential to protect rabbit and human RBCs and human PMNs. For the protection of rabbit RBCs, HlaH35L toxoid-IgG was also required, whereas for human PMNs, LukS-IgG and LukAE323AB-IgG were essential too. When the toxin/toxoid antigens HlgA, LukS-PV, HlaH35L, and LukAE323AB were used to immunize rabbits, they increased rabbit survival; however, they did not block staphylococcal abscess formation in kidneys. Based on these results, we proposed that the combination of HlgA, LukS, HlaH35L, and LukAE323AB is the optimal vaccine component to protect human RBCs and PMNs from staphylococcal PFTs. We also concluded that a successful S. aureus vaccine requires not only those toxin antigens but also other antigens that can induce immune responses blocking staphylococcal colonization.

Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. METHODS: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. RESULTS: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. CONCLUSIONS: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

Efficacy of Active Immunization With Attenuated α-Hemolysin and Panton-Valentine Leukocidin in a Rabbit Model of Staphylococcus aureus Necrotizing Pneumonia.

Staphylococcus aureus is a common pathogen causing infections in humans with various degrees of severity, with pneumonia being one of the most severe infections. In as much as staphylococcal pneumonia is a disease driven in large part by α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL), we evaluated whether active immunization with attenuated forms of Hla (HlaH35L/H48L) alone, PVL components (LukS-PVT28F/K97A/S209A and LukF-PVK102A) alone, or combination of all 3 toxoids could prevent lethal challenge in a rabbit model of necrotizing pneumonia caused by the USA300 community-associated methicillin-resistant S. aureus (MRSA). Rabbits vaccinated with Hla toxoid alone or PVL components alone were only partially protected against lethal pneumonia, whereas those vaccinated with all 3 toxoids had 100% protection against lethality. Vaccine-mediated protection correlated with induction of polyclonal antibody response that neutralized not only α-hemolysin and PVL, but also other related toxins, produced by USA300 and other epidemic MRSA clones.

Deimmunizing substitutions in Pseudomonas exotoxin domain III perturb antigen processing without eliminating T-cell epitopes.

Effective adaptive immune responses depend on activation of CD4+ T cells via the presentation of antigen peptides in the context of major histocompatibility complex (MHC) class II. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity of peptides that are presented to T cells. A recombinant immunotoxin, comprising exotoxin A domain III (PE-III) from Pseudomonas aeruginosa and a cancer-specific antibody fragment, has been developed to manage cancer, but its effectiveness is limited by the induction of neutralizing antibodies. Here, we observed that this immunogenicity is substantially reduced by substituting six residues within PE-III. Although these substitutions targeted T-cell epitopes, we demonstrate that reduced conformational stability and protease resistance were responsible for the reduced antibody titer. Analysis of mouse T-cell responses coupled with biophysical studies on single-substitution versions of PE-III suggested that modest but comprehensible changes in T-cell priming can dramatically perturb antibody production. The most strongly responsive PE-III epitope was well-predicted by a structure-based algorithm. In summary, single-residue substitutions can drastically alter the processing and immunogenicity of PE-III but have only modest effects on CD4+ T-cell priming in mice. Our findings highlight the importance of structure-based processing constraints for accurate epitope prediction.

Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes.

Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling through the Interleukin-1 Receptor.

Group A Streptococcus (GAS) is the etiologic agent of numerous high-morbidity and high-mortality diseases. Infections are typically highly proinflammatory. During the invasive infection necrotizing fasciitis, this is in part due to the GAS protease SpeB directly activating interleukin-1ß (IL-1ß) independent of the canonical inflammasome pathway. The upper respiratory tract is the primary site for GAS colonization, infection, and transmission, but the host-pathogen interactions at this site are still largely unknown. We found that in the murine nasopharynx, SpeB enhanced IL-1ß-mediated inflammation and the chemotaxis of neutrophils. However, neutrophilic inflammation did not restrict infection and instead promoted GAS replication and disease. Inhibiting IL-1ß or depleting neutrophils, which both promote invasive infection, prevented GAS infection of the nasopharynx. Mice pretreated with penicillin became more susceptible to GAS challenge, and this reversed the attenuation from neutralization or depletion of IL-1ß, neutrophils, or SpeB. Collectively, our results suggest that SpeB is essential to activate an IL-1ß-driven neutrophil response. Unlike during invasive tissue infections, this is beneficial in the upper respiratory tract because it disrupts colonization resistance mediated by the microbiota. This provides experimental evidence that the notable inflammation of strep throat, which presents with significant swelling, pain, and neutrophil influx, is not an ineffectual immune response but rather is a GAS-directed remodeling of this niche for its pathogenic benefit.

Regulation of toxic shock syndrome toxin-1 by the accessory gene regulator in Staphylococcus aureus is mediated by the repressor of toxins.

Toxic shock syndrome toxin-1 (TSST-1) is a superantigen (SAg) produced by Staphylococcus aureus thought to be responsible for essentially all cases of menstrual-associated toxic shock syndrome (TSS). As a potent exotoxin, it is not surprising that S. aureus has evolved multiple systems to control expression of TSST-1. Although the accessory gene regulator (Agr) system is recognized to enhance TSST-1 expression, how Agr regulates TSST-1 is unclear. Using an agr-null mutant, complementation experiments demonstrated that Agr controls TSST-1 expression through the activity of the RNAIII effector molecule. RNAIII can repress translation of the repressor of toxins (Rot) regulator, and deletion of rot increased expression of TSST-1 during the exponential phase of growth. Deletion of agr did not affect rot transcription, but did result in overexpression of the Rot protein, and Rot was also shown to bind and positively regulate the rot promoter. Overexpression of Rot dramatically repressed TSST-1, and Rot bound directly to the TSST-1 promoter. Deletion of both agr and rot in S. aureus returned TSST-1 expression to wild-type levels. This work demonstrates that Agr, although widely considered to be an inducer of TSST-1, has evolved in combination with Rot, to restrict the expression of this potent SAg.

Low-Dose Methotrexate Prevents Primary and Secondary Humoral Immune Responses and Induces Immune Tolerance to a Recombinant Immunotoxin.

Recombinant immunotoxins (RITs) are chimeric proteins being developed for cancer treatment. They are composed of an Ab fragment that targets a cancer Ag and a cytotoxic portion of Pseudomonas exotoxin A. They are effective for patients with hematologic malignancies with defective immunity, but their efficacy against solid tumors is limited by anti-drug Ab (ADA) responses in immune-competent patients. Pre-existing Abs or immune memory owing to previous toxin exposure represent additional hurdles because they induce rapid and strong ADA responses. Here, we evaluated the efficacy of methotrexate (MTX) to prevent ADA formation against the mesothelin-targeting RIT LMB-100 in naive mice and in mice with pre-existing Abs. We found that low-dose MTX combined with LMB-100 completely suppressed the formation of ADAs in a dose- and frequency-dependent manner. Suppression of the immune response restored blood levels of LMB-100 and prevented its neutralization. Furthermore, combination of MTX with LMB-100 did not compromise the immune response against a second Ag given after stopping MTX, indicating specific immune tolerance. Adoptive transfer of splenocytes suppressed Ab responses to LMB-100 in recipient mice, indicating a durable immune tolerance. We conclude that combination of MTX and LMB-100 is effective at preventing immune responses in a durable, Ag-specific manner. We propose combining low-dose MTX in immune-competent cancer patients receiving RIT therapy to prevent immunogenicity. This approach could be applied to other immunogenic therapeutic agents and to proteins for which there is pre-existing immunity.

SpeS: A Novel Superantigen and Its Potential as a Vaccine Adjuvant against Strangles.

Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining Antigen-Presenting Cell (APC)-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen-presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses.

Immunogenicity of therapeutic recombinant immunotoxins.

Recombinant immunotoxins (RITs) are chimeric proteins designed to treat cancer. They are made up of an Fv or Fab that targets an antigen on a cancer cell fused to a 38-kDa portion of Pseudomonas exotoxin A (PE38). Because PE38 is a bacterial protein, it is highly immunogenic in patients with solid tumors that have normal immune systems, but much less immunogenic in patients with hematologic malignancies where the immune system is suppressed. RITs have shown efficacy in refractory hairy cell leukemia and in some children with acute lymphoblastic leukemia, but have been much less effective in solid tumors, because neutralizing antibodies develop and prevent additional treatment cycles. In this paper we will (i) review data from clinical trials describing the immunogenicity of PE38 in different patient populations; (ii) review results from clinical trials using different immunosuppressive drugs; and (iii) describe our efforts to make new less-immunogenic RITs by identifying and removing T- and B-cell epitopes to hide the RIT from the immune system.

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death and reduced immunoglobulin release.

Streptococcal pyrogenic exotoxin (Spe) A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage to Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leucocyte profile of tonsil is unique, the impact of SpeA production on human tonsil cell function was investigated. Human tonsil cells from routine tonsillectomy were co-incubated with purified streptococcal superantigens or culture supernatants from isogenic streptococcal isolates, differing only in superantigen production. Tonsil cell proliferation was quantified by tritiated thymidine incorporation, and cell surface characteristics assessed by flow cytometry. Soluble mediators including immunoglobulin were measured using enzyme-linked immunosorbent assay. Tonsil T cells proliferated in response to SpeA and demonstrated typical release of proinflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7 days. In contrast, marked B cell apoptosis and abrogation of total immunoglobulin (Ig)A, IgM, and IgG production occurred in the presence of SpeA and other superantigens. In SpeA-stimulated cultures, T follicular helper (Tfh) cells showed a reduction in C-X-C chemokine receptor (CXCR)5 (CD185) expression, but up-regulation of OX40 (CD134) and inducible T cell co-stimulator (ICOS) (CD278) expression. The phenotypical change in the Tfh population was associated with impaired chemotactic response to CXCL13. SpeA and other superantigens cause dysregulated tonsil immune function, driving T cells from Tfh to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a probable survival advantage.

Phase 1 study of the anti-CD22 immunotoxin moxetumomab pasudotox for childhood acute lymphoblastic leukemia.

Novel therapies are needed to overcome chemotherapy resistance for children with relapsed/refractory acute lymphoblastic leukemia (ALL). Moxetumomab pasudotox is a recombinant anti-CD22 immunotoxin. A multicenter phase 1 study was conducted to determine the maximum-tolerated cumulative dose (MTCD) and evaluate safety, activity, pharmacokinetics, and immunogenicity of moxetumomab pasudotox in children, adolescents, and young adults with ALL (N = 55). Moxetumomab pasudotox was administered as a 30-minute IV infusion at doses of 5 to 50 µg/kg every other day for 6 (cohorts A and B) or 10 (cohort C) doses in 21-day cycles. Cohorts B and C received dexamethasone prophylaxis against capillary leak syndrome (CLS). The most common treatment-related adverse events were reversible weight gain, hepatic transaminase elevation, and hypoalbuminemia. Dose-limiting CLS occurred in 2 of 4 patients receiving 30 µg/kg of moxetumomab pasudotox every other day for 6 doses. Incorporation of dexamethasone prevented further dose-limiting CLS. Six of 14 patients receiving 50 µg/kg of moxetumomab pasudotox for 10 doses developed hemolytic uremic syndrome (HUS), thrombotic microangiopathy (TMA), or HUS-like events, exceeding the MTCD. Treatment expansion at 40 µg/kg for 10 doses (n = 11) exceeded the MTCD because of 2 HUS/TMA/HUS-like events. Dose level 6B (ie, 50 µg/kg × 6 doses) was the MTCD, selected as the recommended phase 2 dose. Among 47 evaluable patients, an objective response rate of 32% was observed, including 11 (23%) composite complete responses, 5 of which were minimal residual disease negative by flow cytometry. Moxetumomab pasudotox showed a manageable safety profile and evidence of activity in relapsed or refractory childhood ALL. This trial was registered at www.clinicaltrials.gov as #NCT00659425.

Investigation of Fibronectin Binding Protein (FBP) and Panton Valentine Leukocidin (PVL) Viulance Factors in Clinical Methicillin Sensitive and Resistant Staphylococcus Aureus Strains.

BACKGROUND: We aimed to investigate the frequency of fibronectin binding protein (FBP), which is part of the first step of adhesion, and Panton-Valentine leukocidin (PVL) toxin, which contributes to the destruction of host leukocytes and tissue necrosis, in clinical S. aureus strains. METHODS: One hundred S. aureus strains were included in the study and distributed as follows; 33 from skinwound swabs and catheter tips (SWCT), 33 from body fluid and secretion specimens (BSFS) such as tracheal aspirate, sputum, and pleural effusion fluid, 18 from tissue biopsy specimens (TBS), 10 specimens from blood, and related specimens (BRS) such as bone marrow, and cerebral spinal fluid, and six specimens from mucosal membrane of pharynx, nose, and vagina (MMS). Methicillin resistance was tested by disk diffusion method. mecA (methicillin resistance coded gene), pvl and fnbA genes were investigated by using a PCR method. RESULTS: Thirty-seven strains (37.0%) were identified as methicillin resistant S. aureus (MRSA) and 63 (63.0%) as methicillin susceptible S. aureus (MSSA) strains. fnbA was more frequent in S. aureus isolates of MMSs (100.0%); followed by BRSs (80.0%), SWCTs (78.8%), TBS (72.3%), and BSFs (66.7%), whereas pvl gene was more frequent in isolates of BRS (60.0%), followed by TBSs (50.0%), SWCTs (33.4%), BSFs (30.3%), and MMSs (16.7%). fnbA existed in 85.7% of MSSA and 56.8% of MRSA in contrast to pvl, which was more frequent in MRSA (70.3%) than those of MSSA strains (17.4%). These differences were statistically significant (p < 0.05). CONCLUSIONS: Our different clinical specimens contained a high rate of fnbA (75.0%) and low-moderate frequency of pvl (37.0%). fnbA was most frequent in S. aureus of MMSs, followed by BRSs, and SWCTs, whereas pvl was ex-isted in high proportion in S. aureus of BRSs, followed by TBSs, and SWCTs. Presence of PVL in a high proportion in MRSA strains of superfical specimens such SWCT (24.4%) and deeper serious specimens such as BRS (16.3%) compared to MSSA strains from the same specimens, 3.2% and 0%, respectively, have shown that MRSA infections still threatens patients' lives and control of their spread is urgently needed.

Streptococcal Pyrogenic Exotoxin A-Stimulated Monocytes Mediate Regulatory T-Cell Accumulation through PD-L1 and Kynurenine.

Bacterial superantigens (SAgs) are exotoxins that promote a fulminant activation of the immune system. The subsequent intense release of inflammatory cytokines often results in hypotension, shock, and organ failure with high mortality rates. In the current paradigm, the direct and simultaneous binding of SAgs with T-cell receptor (TCR)-bearing Vß regions and conserved structures on major histocompatibility complex class II (MHC class II) on antigen-presenting cells (APCs) induces the activation of both cell types. However, by crosslinking MHC class II molecules, APCs can be activated by SAgs independently of T lymphocytes. Recently, we showed that streptococcal pyrogenic exotoxin A (SPEA) of Streptococcus pyogenes stimulates an immunogenic APC phenotype with upregulated costimulatory molecules and inflammatory cytokines. Additionally, we revealed that SPEA triggers immunosuppressive programs in monocytes that facilitate the accumulation of regulatory T cells (Tregs) in in vitro monocyte/CD4+ T-cell cocultures. Immunosuppressive factors include anti-inflammatory interleukin 10 (IL-10), co-inhibitory surface molecule programmed cell death 1 ligand 1 (PD-L1), and the inhibitory indoleamine 2,3-dioxygenase (IDO)/kynurenine effector system. In the present study, we investigated the underlying mechanism of SPEA-stimulated monocyte-mediated accumulation of Tregs. Blood-derived monocytes from healthy donors were stimulated with SPEA for 48 h (SPEA-monocytes). For the evaluation of SPEA-monocyte-mediated modulation of CD4+ T lymphocytes, SPEA was removed from the culture through extensive washing of cells before adding allogeneic CD3/CD28-activated T cells. Results: In coculture with allogeneic CD4+ T cells, SPEA-monocytes mediate apoptosis of CD4+Foxp3- lymphocytes and accumulation of CD4+Foxp3+ Tregs. PD-L1 and kynurenine are critically involved in the mediated cell death because blocking both factors diminished apoptosis and decreased the proportion of the CD25+/Foxp3+ Treg subpopulation significantly. Upregulation of PD-L1 and kynurenine as well as SPEA-monocyte-mediated effects on T cells depend on inflammatory IL-1ß. Our study shows that monocytes activated by SPEA mediate apoptosis of CD4+Foxp3- T effector cells through PD-L1 and kynurenine. CD4+Foxp3+ T cells are resistant to apoptosis and accumulate in SPEA-monocyte/CD4+ T-cell coculture.

HER2-Specific Targeted Toxin DARPin-LoPE: Immunogenicity and Antitumor Effect on Intraperitoneal Ovarian Cancer Xenograft Model.

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.