Designing of a chimeric protein contains StxB, intimin and EscC against toxicity and adherence of enterohemorrhagic Escherichia coli O157:H7 and evaluation of serum antibody titers against it.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC.

Immunization against Vibrio cholerae, ETEC, and EHEC with chitosan nanoparticle containing LSC chimeric protein.

INTRODUCTION: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity. METHODS: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse. RESULTS: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins. CONCLUSION: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria.

Antibodies to Shiga toxins in Brazilian cattle.

Cattle are considered a reservoir of Shiga toxin-producing Escherichia coli (STEC). There is no information about the presence of antibodies against Shiga toxins in Brazilian bovine serum. Using ELISA, all sera tested showed antibodies against the two main STEC virulence factors; Stx1 and Stx2. Neutralizing antibodies against Stx1 and/or Stx2 were detected in all but one serum. In conclusion, our results indicated that these animals had been exposed to STEC producing both toxins.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1ß and Apoptotic Cell Death.

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer.

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)-based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8(+) T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8(+) T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials.

Detection of shiga toxins by lateral flow assay.

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

A globotetraosylceramide (Gb4) receptor-based ELISA for quantitative detection of Shiga toxin 2e.

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20-2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.

Advances in the development of enterohemorrhagic Escherichia coli vaccines using murine models of infection.

Enterohemorrhagic Escherichia coli (EHEC) strains are food borne pathogens with importance in public health. EHEC colonizes the large intestine and causes diarrhea, hemorrhagic colitis and in some cases, life-threatening hemolytic-uremic syndrome (HUS) due to the production of Shiga toxins (Stx). The lack of effective clinical treatment, sequelae after infection and mortality rate in humans supports the urgent need of prophylactic approaches, such as development of vaccines. Shedding from cattle, the main EHEC reservoir and considered the principal food contamination source, has prompted the development of licensed vaccines that reduce EHEC colonization in ruminants. Although murine models do not fully recapitulate human infection, they are commonly used to evaluate EHEC vaccines and the immune/protective responses elicited in the host. Mice susceptibility differs depending of the EHEC inoculums; displaying different mortality rates and Stx-mediated renal damage. Therefore, several experimental protocols have being pursued in this model to develop EHEC-specific vaccines. Recent candidate vaccines evaluated include those composed of virulence factors alone or as fused-subunits, DNA-based, attenuated bacteria and bacterial ghosts. In this review, we summarize progress in the design and testing of EHEC vaccines and the use of different strategies for the evaluation of novel EHEC vaccines in the murine model.

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells.

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), macrophage inflammatory protein-1α/ß (MIP-1α/ß), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groß. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Functional assay for shiga-like toxin via detection by antibody capture and multivalent galabiose binding.

A functional detection assay was developed for Escherichia coli secreted shiga-like toxin based on antibody capture and visualization with a multivalent galabiose ligand. It was possible to detect verotoxin in medically relevant E. coli samples in a dose dependent fashion. This method is a new step towards measuring functional protein levels in complex mixtures, which can be used for diagnostic purposes in a clinical setting.

Enterohemorrhagic Escherichia coli O157:H7 Shiga toxins inhibit gamma interferon-mediated cellular activation.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

Management of hemolytic uremic syndrome.

2011 has been a special year for hemolytic uremic syndrome (HUS): on the one hand, the dramatic epidemic of Shiga toxin producing E. coli -associated HUS in Germany brought the disease to the attention of the general population, on the other hand it has been the year when eculizumab, the first complement blocker available for clinical practice, was demonstrated as the potential new standard of care for atypical HUS. Here we review the therapeutic options presently available for the various forms of hemolytic uremic syndrome and show how recent knowledge has changed the therapeutic approach and prognosis of atypical HUS.

Live attenuated Shigella dysenteriae type 1 vaccine strains overexpressing shiga toxin B subunit.

Shigella dysenteriae serotype 1 (S. dysenteriae 1) is unique among the Shigella species and serotypes in the expression of Shiga toxin which contributes to more severe disease sequelae and the ability to cause explosive outbreaks and pandemics. S. dysenteriae 1 shares characteristics with other Shigella species, including the capability of causing clinical illness with a very low inoculum (10 to 100 CFU) and resistance to multiple antibiotics, underscoring the need for efficacious vaccines and therapeutics. Following the demonstration of the successful attenuating capacity of deletion mutations in the guaBA operon in S. flexneri 2a vaccine strains in clinical studies, we developed a series of S. dysenteriae 1 vaccine candidates containing the fundamental attenuating mutation in guaBA. All strains are devoid of Shiga toxin activity by specific deletion of the gene encoding the StxA subunit, which encodes enzymatic activity. The StxB subunit was overexpressed in several derivatives by either plasmid-based constructs or chromosomal manipulation to include a strong promoter. All strains are attenuated for growth in vitro in the HeLa cell assay and for plaque formation and were safe in the Serény test and immunogenic in the guinea pigs. Each strain induced robust serum and mucosal anti-S. dysenteriae 1 lipopolysaccharide (LPS) responses and protected against wild-type challenge. Two strains engineered to overexpress StxB induced high titers of Shiga toxin neutralizing antibodies. These candidates demonstrate the potential for a live attenuated vaccine to protect against disease caused by S. dysenteriae 1 and potentially to protect against the toxic effects of other Shiga toxin 1-expressing pathogens.

Novel fusion protein protects against adherence and toxicity of enterohemorrhagic Escherichia coli O157:H7 in mice.

Infection with Escherichia coli (E. coli) O157:H7 may develop into bloody diarrhea, or hemorrhagic uremic syndrome (HUS), which usually causes kidney failure or even death. Considered as the pathogenesis mechanism of E. coli O157:H7 infection, attachment or adhesion that is directly mediated by intimin is the first step of E. coli O157:H7 interaction with its host, and all these serious sequelae are mainly due to Shiga toxins (Stxs) released by E. coli O157:H7. In this study, a novel SSI fusion protein that contains the critical toxin-antigens Stx2B and Stx1B, and the critical adhesion-antigen fragment Int281 was constructed. The protein induced complete immune protection, with both anti-toxin and anti-adhesion effects. The dominant increase in IgG1 and the high level of Th2-typical cytokine (IL-4 and IL-10) expression showed that SSI significantly induced Th2-mediated humoral immune response. In the mouse model, the SSI fusion protein not only elicited neutralizing antibodies against both Stx1 and Stx2 toxins, but also induced a high level of anti-adhesion antibodies. The SSI-immunized mice did not show any pathologic changes. SSI provides evident protection with two-time immunization against a highly lethal dose of E. coli O157:H7.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.

Antibody response to Shiga toxins in Argentinean children with enteropathic hemolytic uremic syndrome at acute and long-term follow-up periods.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools.

Characterization of rabbit polyclonal sera against recombinant Shiga toxin and its subunits for detection of Stx-producing E. coli.

Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera could also neutralize the cytotoxicity of the shiga toxin on vero cells. The neutralizing ability of the prepared sera was compared for different subunits. The neutralization of toxicity was observed by incubation of raised sera with the rStx or Shiga toxin from wild type strains. The inhibition of cell toxicity was shown by anti-A, anit-B and anti-AB antisera, separately. It was shown that anti-A antibody, more significantly recognized Stx producing strains, comparing to anti-B antibody. These sera from immunized rabbits were also used as specific antibodies in Enzyme-Linked Immunosorbant Assay (ELISA) for detection of Shiga toxin. It was demonstrated that the raised antibodies especially antibody against A subunit could be a useful tool for immunological diagnosis of STEC induced infection.

Assessment of immune response of the B subunit of Shiga toxin fused to AAF adhesin of enteroaggregative Escherichia coli.

Shiga toxin is a member of AB toxin family and is composed of an A subunit which mediated toxicity and a homopentameric protein responsible for toxin binding and internalization into target cells. Another group of diarrheagenic Escherichia coli, enteroaggregative E. coli (EAEC) is a group of E. coli with aggregative adherence to epithelial cells, which play an important role in its pathogenesis. In the present investigation, the immune response of recombinant hybrid peptide composed of B subunit of Shiga toxin (StxB) and Aggregative Adherence Fimbriae (AAF) of EAEC (B-AAF/I, B-AAF/II) that elicited protective response was further characterized. The assessment of IgG subclasses (IgG1 and IgG2a) and cytokine production by these peptides indicated that although the hybrid peptides could induce immune response, but two adhesins behave differently in this regard. Lymphocyte proliferation assay and IFN-γ production were highly significant for B-AAF/II. Overall, based on the data obtained from this study it seems that mixed population of Th1-Th2 type of immune responses were induced by these hybrid peptides, which probably lead to observed protective response. In the present study, it is shown that the two hybrid peptides i.e. B-AAF/I and B-AAF/II, could be a promising strategy to make more effective and powerful vaccine.

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria.

AIMS: The Shiga-like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. METHODS AND RESULTS: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non-Stx1-producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. CONCLUSIONS: The anti-STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report regarding the usage of anti-STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.

A recombinant hybrid peptide composed of AAF adhesin of enteroaggregative Escherichia coli and Shiga toxin B subunit elicits protective immune response in mice.

Shiga toxin producing Escherichia coli (STEC) are a group of diarrheagenic Escherichia coli (E. coli) whereby Shiga toxin is the main virulence factor. It is composed of an A subunit, which mediates toxicity, and a B subunit (StxB), which is a nontoxic homopentameric protein responsible for toxin binding and internalization into target cells by interacting with the glycolipid, globotriaosylceramide (Gb3). Enteroaggregative Escherichia coli (EAEC) are a group of E. coli with aggregative adherence to epithelial cells, which play an important role in its pathogenesis. EAEC are the cause of diarrhea in developing countries and in the developed world. Aggregative adherence fimbria (AAF) of EAEC represents the adhesin that confers the presence of aggregative adherence (AA) phenotype on EAEC strains. The gene encoding non-toxic B subunit of Shiga toxin (StxB) was coupled to aggregative adherence fimbriae (AAF) of the EAEC structural gene. The resulting polypeptides (B-AAF/I, B-AAF/II) were designed to elicit immune response in immunized mice with recombinant peptides. The antibody, hence obtained, inhibited the adherence of prototype EAEC strains to HeLa cells and, on the other hand, protected the immunized mice against a lethal dose of Shiga toxin. Therefore, this promising data could indicate that this kind of polypeptide strategy is a good candidate for any probable vaccine design against diarrheal infection.