RESUMEN
The rectal-anal junction (RAJ) is the major colonization site of Shiga toxin-producing Escherichia coli (STEC) O157 in beef cattle, leading to transmission of this foodborne pathogen from farms to food chains. To date, there is limited understanding regarding whether the mucosa-attached microbiome has a profound impact on host-STEC interactions. In this study, the active RAJ mucosa-attached microbiota and its potential role in host immunity-STEC commensal interactions were investigated using RAJ mucosal biopsies collected from calves orally challenged with two STEC O157 strains with or without functional stx2a (stx2a+ or stx2a-). The results revealed that shifts of microbial diversity, topology, and assembly patterns were subjected to stx2a production post-challenge and Paeniclostridium and Gallibacterium were the keystone taxa for both microbial interactions and assembly. Additional mucosal transcriptome profiling showed stx2a-dependent host immune responses (i.e. B- and T-cell signaling and antigen processing and presentation) post-challenge. Further integrated analysis revealed that mucosa-attached beneficial microbes (i.e. Provotella, Faecalibacterium, and Dorea) interacted with host immune genes pre-challenge to maintain host homeostasis; however, opportunistic pathogenic microbes (i.e. Paeniclostridium) could interact with host immune genes after the STEC O157 colonization and interactions were stx2a-dependent. Furthermore, predicted bacterial functions involved in pathogen (O157 and Paeniclostridium) colonization and metabolism were related to host immunity. These findings suggest that during pathogen colonization, host-microbe interactions could shift from beneficial to opportunistic pathogenic bacteria driven and be dependent on the production of particular virulence factors, highlighting the potential regulatory role of mucosa-attached microbiota in affecting pathogen-commensal host interactions in calves with STEC O157 infection.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli O157 , Mucosa Intestinal , Recto , Animales , Escherichia coli O157/inmunología , Escherichia coli O157/genética , Recto/microbiología , Bovinos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/veterinaria , Mucosa Intestinal/microbiología , Mucosa Intestinal/inmunología , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/inmunología , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Interacciones Microbiota-Huesped/inmunología , Toxina Shiga II/genética , Toxina Shiga II/inmunologíaRESUMEN
Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Síndrome Hemolítico-Urémico/prevención & control , Fragmentos Fab de Inmunoglobulinas/inmunología , Toxina Shiga II/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Apoptosis/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Proteínas Recombinantes , Toxina Shiga I/inmunología , Toxina Shiga I/toxicidad , Toxina Shiga II/toxicidad , Escherichia coli Shiga-Toxigénica/inmunología , Células VeroRESUMEN
Inflammatory caspase-dependent cytosolic lipopolysaccharide (LPS) sensing is a critical arm of host defense against bacteria. How pathogens overcome this pathway to establish infections is largely unknown. Enterohemorrhagic Escherichia coli (EHEC) is a clinically important human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. We found that a bacteriophage-encoded virulence factor of EHEC, Shiga toxin (Stx), suppresses caspase-11-mediated activation of the cytosolic LPS sensing pathway. Stx was essential and sufficient to inhibit pyroptosis and interleukin-1 (IL-1) responses elicited specifically by cytosolic LPS. The catalytic activity of Stx was necessary for suppression of inflammasome responses. Stx impairment of inflammasome responses to cytosolic LPS occurs at the level of gasdermin D activation. Stx also suppresses inflammasome responses in vivo after LPS challenge and bacterial infection. Overall, this study assigns a previously undescribed inflammasome-subversive function to a well-known bacterial toxin, Stx, and reveals a new phage protein-based pathogen blockade of cytosolic immune surveillance.
Asunto(s)
Escherichia coli Enterohemorrágica/patogenicidad , Infecciones por Escherichia coli/inmunología , Inflamasomas/inmunología , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Animales , Bacteriófagos/inmunología , Bacteriófagos/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Chlorocebus aethiops , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Modelos Animales de Enfermedad , Escherichia coli Enterohemorrágica/inmunología , Escherichia coli Enterohemorrágica/virología , Infecciones por Escherichia coli/microbiología , Femenino , Humanos , Vigilancia Inmunológica , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfato/metabolismo , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Células Vero , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world as well as in the United States. Current detection methods have limitation to implement for rapid field-deployable detection with high volume of samples that are needed for regulatory purposes. Surface plasmon resonance imaging (SPRi) has proved to achieve rapid and label-free screening of multiple pathogens simultaneously, so it was evaluated in this work for the detection of Shiga toxins (Stx1a and Stx2a toxoids were used as the less toxic alternatives to Stx1 and Stx2, respectively). Multiple antibodies (Stx1pAb, Stx1-1mAb, Stx1-2mAb, Stx1d-3mAb, Stx1e-4mAb, Stx2pAb, Stx2-1mAb, Stx2-2mAb, and Stx2-10mAb) were spotted one by one by programed microarrayer, on the same high-throughput biochip with 50-nm gold film through multiple crosslinking and blocking steps to improve the orientation of antibodies on the biochip surface. Shiga toxins were detected based on the SPRi signal difference (ΔR) between immobilized testing antibodies and immunoglobulin G (IgG) control. Among the antibodies tested, Stx1pAb showed the highest sensitivity for Stx1 toxoid, with the limit of detection (LOD) of 50 ng/mL and detection time of 20 min. Both Stx2-1mAb and Stx2-2mAb exhibited high sensitivity for Stx2 toxoid. Furthermore, gold nanoparticles (GNPs) were used to amplify the SPRi signals of monoclonal antibodies in a sandwich platform. The LOD reached the level of picogram (pg)/mL with the help of GNP-antibody conjugate. This result proved that SPRi biochip with selected antibodies has the potential for rapid, high-throughput and multiplex detection of Shiga toxins.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ensayos Analíticos de Alto Rendimiento , Inmunoensayo , Análisis por Micromatrices , Toxina Shiga I/análisis , Toxina Shiga II/análisis , Resonancia por Plasmón de Superficie , Especificidad de Anticuerpos , Oro/química , Límite de Detección , Nanopartículas del Metal , Reproducibilidad de los Resultados , Toxina Shiga I/inmunología , Toxina Shiga II/inmunologíaRESUMEN
Two types of lateral flow immunochromatographic test strips (LFITS) using gold nanoparticles and fluorescent CdTe quantum dots (QDs) as signal labels, respectively, were developed for Shiga toxin type II (STX2) assays. Under optimal conditions, the corresponding visual detection limits were 25 ng mL-1 and 5 ng mL-1, respectively. The test results of gold based LFITS can be recognized directly by the naked eye, whereas the visualized results of CdTe QDs based LFITS can be observed by the aid of a UV lamp. Both assays showed good specificity and stability. The inexpensive LFITS were promising for the rapid clinical detection of STX2.
Asunto(s)
Cromatografía de Afinidad/métodos , Colorimetría/métodos , Fluorometría/métodos , Tiras Reactivas , Toxina Shiga II/análisis , Anticuerpos/inmunología , Compuestos de Cadmio/química , Cromatografía de Afinidad/instrumentación , Colorimetría/instrumentación , Colorantes Fluorescentes/química , Fluorometría/instrumentación , Oro/química , Límite de Detección , Nanopartículas del Metal/química , Puntos Cuánticos/química , Toxina Shiga II/inmunología , Telurio/químicaRESUMEN
There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Animales , Dominio Catalítico/genética , Línea Celular , Chlorocebus aethiops , Infecciones por Escherichia coli/patología , Citometría de Flujo , Unión Proteica/fisiología , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Células VeroRESUMEN
Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB-transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB-lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB-lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB-lettuce is a promising first oral vaccine candidate against ED.
Asunto(s)
Animales Recién Nacidos , Vacunas Bacterianas/administración & dosificación , Edematosis Porcina/etiología , Edematosis Porcina/prevención & control , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/veterinaria , Lactuca , Toxina Shiga II/inmunología , Escherichia coli Shiga-Toxigénica , Porcinos , Destete , Administración Oral , Animales , Escherichia coli Shiga-Toxigénica/patogenicidad , VirulenciaRESUMEN
Food poisonings caused by Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) occur frequently worldwide; however, no vaccine is currently available. Therefore, we aimed to develop a bivalent vaccine against C. perfringens and STEC infections. Although it has been considered that the C-terminal region of C. perfringens enterotoxin (C-CPE) could be a good vaccine antigen to block the binding to its receptor, it was insufficient for induction of a protective immune response because of the low antigenicity. However, the fusion of C-CPE with Stx2 B subunit (Stx2B) augmented the antigenicity of C-CPE without affecting the antigenicity of Stx2B. Indeed, high levels of C-CPE-specific neutralizing IgG were found in the serum of mice immunized with the fusion protein Stx2B-C-CPE. Additionally, comparable and substantial levels of Stx2B-specific neutralizing IgG were induced in mice receiving Stx2B-C-CPE or Stx2B alone. These antibody responses against C-CPE and Stx2B lasted for at least 48 weeks, which were sufficient for protective immunity in vitro and in vivo, indicating that Stx2B-C-CPE could induce long-term protective immunity. As an underlying mechanism, ex vivo stimulation with Stx2B, but not with C-CPE, induced cytokine production from splenic T cells collected from mice immunized with Stx2B-C-CPE, suggesting that Stx2B-specific, but not C-CPE-specific, T cells were induced by the immunization with Stx2B-C-CPE and plausibly promoted immunoglobulin class switching of both Stx2B- and C-CPE-specific B cells from IgM to IgG. These findings collectively indicate that Stx2B-C-CPE is a T-cell-antigen-supplement-type bivalent vaccine, which could be an efficient against C. perfringens and STEC infections.
Asunto(s)
Clostridium perfringens/inmunología , Enterotoxinas/inmunología , Escherichia coli/inmunología , Enfermedades Transmitidas por los Alimentos/inmunología , Inmunogenicidad Vacunal/inmunología , Toxina Shiga II/inmunología , Vacunas/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.
Asunto(s)
Contaminación de Alimentos/análisis , Inmunoensayo/métodos , Lactuca , Hojas de la Planta/química , Carne Roja/análisis , Toxina Shiga II/análisis , Anticuerpos/inmunología , Límite de Detección , Toxina Shiga II/inmunología , Escherichia coli Shiga-Toxigénica/metabolismoRESUMEN
Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57â¯Bâ¯l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (pâ¯<â¯0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (pâ¯<â¯0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (pâ¯<â¯0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection.
Asunto(s)
Dieta/efectos adversos , Síndrome Hemolítico-Urémico/etiología , Mediadores de Inflamación , Riñón/efectos de los fármacos , Obesidad/complicaciones , Toxina Shiga II/toxicidad , Animales , Glucemia , Quimiocina CCL2/metabolismo , Creatinina/sangre , Citocinas/sangre , Modelos Animales de Enfermedad , Escherichia coli , Femenino , Síndrome Hemolítico-Urémico/inducido químicamente , Síndrome Hemolítico-Urémico/patología , Receptor Celular 1 del Virus de la Hepatitis A , Inflamación , Interleucina-1alfa/sangre , Interleucina-1beta/metabolismo , Interleucina-6/sangre , Riñón/patología , Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Necrosis , Neutrófilos/efectos de los fármacos , Toxina Shiga II/inmunología , Factor de Necrosis Tumoral alfa/sangre , Aumento de PesoRESUMEN
Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E.coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E.coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspBâ¯+â¯IntC280, with EspBâ¯+â¯IntC280â¯+â¯BLS-Stx2B, or kept as controls. At 24â¯days post vaccination calves were challenged with E.coli O157:H7. Shedding of E.coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15â¯days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E.coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E.coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds.
Asunto(s)
Adhesinas Bacterianas/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Infecciones por Escherichia coli/veterinaria , Escherichia coli O157/inmunología , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Síndrome Hemolítico-Urémico/prevención & control , Toxina Shiga II/inmunología , Zoonosis/prevención & control , Adhesinas Bacterianas/genética , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Proteínas de la Membrana Bacteriana Externa/genética , Derrame de Bacterias , Bovinos , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/genética , Vacunas contra Escherichia coli/inmunología , Vacunas contra Escherichia coli/uso terapéutico , Heces/microbiología , Humanos , Inmunidad Humoral/inmunología , Mucosa Intestinal/inmunología , Masculino , Toxina Shiga II/genética , Vacunación/veterinaria , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.
Asunto(s)
Drogas en Investigación , Infecciones por Escherichia coli/tratamiento farmacológico , Síndrome Hemolítico-Urémico/prevención & control , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Toxina Shiga I/antagonistas & inhibidores , Toxina Shiga II/antagonistas & inhibidores , Anticuerpos/inmunología , Argentina , Ensayos Clínicos Fase II como Asunto , Escherichia coli/inmunología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/complicaciones , Síndrome Hemolítico-Urémico/inmunología , Humanos , Toxina Shiga I/inmunología , Toxina Shiga II/inmunologíaRESUMEN
El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.
The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.
Asunto(s)
Humanos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Drogas en Investigación , Toxina Shiga I/antagonistas & inhibidores , Toxina Shiga II/antagonistas & inhibidores , Infecciones por Escherichia coli/tratamiento farmacológico , Síndrome Hemolítico-Urémico/prevención & control , Argentina , Ensayos Clínicos Fase II como Asunto , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Escherichia coli/aislamiento & purificación , Escherichia coli/inmunología , Infecciones por Escherichia coli/complicaciones , Síndrome Hemolítico-Urémico/inmunología , Anticuerpos/inmunologíaRESUMEN
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. RESULTS: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113-rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. CONCLUSIONS: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.
Asunto(s)
Anticuerpos Neutralizantes , Proteínas Recombinantes , Toxina Shiga II , Anticuerpos de Dominio Único , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/fisiología , Camélidos del Nuevo Mundo/inmunología , Chlorocebus aethiops , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Toxina Shiga II/química , Toxina Shiga II/genética , Toxina Shiga II/inmunología , Toxina Shiga II/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/fisiología , Células VeroRESUMEN
E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli O157/inmunología , Lactoferrina/biosíntesis , Toxina Shiga II/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Especificidad de Anticuerpos/inmunología , Bovinos , Línea Celular Tumoral , Colon/inmunología , Colon/metabolismo , Colon/microbiología , Colon/patología , Escherichia coli O157/patogenicidad , Femenino , Síndrome Hemolítico-Urémico/veterinaria , Humanos , Inmunización , Inmunoglobulina G/inmunología , Masculino , Ratones , Pruebas de Neutralización , Embarazo , RatasRESUMEN
A rapid and sensitive two-step time-resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin-producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu3+ ) chelate was then used as a detector, followed by fluorescence measurements using time-resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1-1000 ng/ml). The intra- and inter-assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2-specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2-specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation-based platform and aid in the accurate and prompt diagnosis of STEC infections.
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Fluoroinmunoensayo/métodos , Toxina Shiga II/análisis , Escherichia coli Shiga-Toxigénica/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Calibración , Ensayo de Inmunoadsorción Enzimática , Europio/química , Fluoroinmunoensayo/instrumentación , Límite de Detección , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Toxina Shiga I/genética , Toxina Shiga II/genética , Toxina Shiga II/inmunología , Escherichia coli Shiga-Toxigénica/genéticaRESUMEN
BACKGROUND AND AIM: An outbreak of Shiga toxin 2 (Stx2) producing enterohemorrhagic and enteroaggregative Escherichia coli O104:H4 infection in May 2011 in Germany caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). We hypothesized that anti-Stx2 IgM or IgG titers might predict HUS development. METHODS: Thirty-two patients infected with enterohemorrhagic Escherichia coli O104:H4 (HUS: n = 23; non-HUS: n = 9) were retrospectively screened for anti-Stx2 IgM/IgG and matched with clinical data regarding HUS development, fever, superinfection, dialysis, neurological symptoms, intensive care, antibiotic treatment, and plasmapheresis. RESULTS: Only HUS patients showed a prominent Stx2-specific humoral response in the early acute phase. Despite a strong trend towards prediction of HUS development, statistical analysis revealed no significant correlation between high IgM/IgG titers and further key clinical parameters such as fever, superinfection, neurological symptoms, antibiotic treatment, and plasmapheresis. CONCLUSIONS: Anti-Stx2 antibodies seem to accompany or even precede HUS development.
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Anticuerpos Antibacterianos/sangre , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/microbiología , Escherichia coli O104/inmunología , Escherichia coli O104/patogenicidad , Síndrome Hemolítico-Urémico/diagnóstico , Síndrome Hemolítico-Urémico/etiología , Toxina Shiga II/inmunología , Reacción de Fase Aguda , Antibacterianos , Biomarcadores/sangre , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/terapia , Fiebre/etiología , Síndrome Hemolítico-Urémico/terapia , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Enfermedades del Sistema Nervioso , Plasmaféresis , Valor Predictivo de las Pruebas , Estudios Retrospectivos , SobreinfecciónRESUMEN
BACKGROUND: In the pathogenicity of porcine edema disease (ED), which is caused by the Escherichia coli-producing F18 and Shiga toxin, F18+ fimbrial adhesins and Shiga toxin 2e (Stx2e) play pivotal roles in the colonization and enterotoxicity of this pathogen. OBJECTIVE: To develop a vaccine candidate against ED by combining three selected antigens of F18+ E. coli. METHODS: Genetically engineered Salmonella Typhimurium (ST) ghosts that express Stx2eB, FedF, and FedA were individually inserted in a ghost plasmid cassette, and the resultant plasmids were transformed into an attenuated ST (JOL912). The individual expression of Stx2eB, FedF, and FedA in JOL912 was validated by using an immunoblotting assay. RESULTS: Immunization of the ghosts in BALB/c mice led to a significant increase in antigen-specific secretory IgA and serum IgG. Significantly marked elevation of the CD3+CD4+ T cell subpopulation and lymphocyte proliferating activity in the primed splenocytes were also observed. Furthermore, mRNA of IL-4 and IFN-γ were highly upregulated in in vitro stimulated splenic T cells. Subsequently, the immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls. CONCLUSION: A mixture of the ghosts expressing these three antigens is a potential vaccine candidate for protection against the porcine edema disease.
Asunto(s)
Adhesinas Bacterianas/inmunología , Edematosis Porcina/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/inmunología , Toxina Shiga II/inmunología , Animales , Edematosis Porcina/inmunología , Edematosis Porcina/microbiología , Femenino , Inmunidad Celular , Inmunidad Humoral , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Salmonella/inmunología , Porcinos , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: Salmonella Typhimurium (S. Typhimurium) inactivated by a regulated autolysis system was genetically engineered to express the homo-pentameric B subunit of Shiga toxin 2e (Stx2eB) on its surface. To prepare a strain able to yield autolyzed Salmonella bearing Stx2eB, the plasmid pJHL184 harboring stx 2eB gene was transformed into the attenuated S. Typhimurium strain, JOL1454. Stx2eB subcloned into the antigen delivery cassette of the plasmid was expressed as fusion protein with the outer membrane protein RESULTS: The expression of Stx2eB fused to the signal peptide in JOL1454 was validated by immunoblot analysis. To determine the immunogenicity of JOL1454, female BALB/c mice were intramuscularly injected with 1 × 108 CFU of the inactivated cells at weeks 0 and 2. Significantly elevated levels of IgG and IgA specific to Stx2eB was observed at weeks 4 and 6 post-immunization (PI) (P <0.05). Proportion of CD3+CD4+ T lymphocyte subpopulation was also significantly augmented in in vivo stimulated splenocytes relative to that in the control group. The increased titers of IgG1 and IgG2a, and of immunomodulatory cytokines indicated that the immunization elicited Th1 and Th2 immune responses. Further, immunomodulatory cytokine genes (IL-6, IL-17A, IL21 and JOL1454) efficiently upregulated in naïve porcine peripheral blood mononuclear cells (PBMCs) pulsed with JOL1454. At week 6 PI, following the challenge with a virulent Stx2e-producing Escherichia coli in the mice, all immunized mice survived whereas approximately 30% of the mice in the control group died. CONCLUSIONS: JOL1454 provided superior immunogenicity and effective protection against challenge with a sublethal dose, which demonstrates its potential as a candidate vaccine against edema disease.
Asunto(s)
Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Salmonella typhimurium/inmunología , Toxina Shiga II/inmunología , Animales , Proliferación Celular , Citocinas/biosíntesis , Infecciones por Escherichia coli/inmunología , Vacunas contra Escherichia coli/genética , Femenino , Inmunidad Celular , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Salmonella typhimurium/genética , Toxina Shiga II/genética , Porcinos , Linfocitos T/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal antibody against Shiga toxin (Stx) 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 108 colony-forming units of EHEC O157:H7 strain EDL933 (Stx1âº, Stx2âº) when 22-24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight). Compared to placebo (n = 10), TMA-15 (n = 35) yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05). The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004) and 71.4% (p = 0.0004), respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2.