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Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions. Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Alvarez, Romina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Alberto, Maria Fabiana. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bruballa, Andrea Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Exeni, Ramon A.. Hospital Municipal del Niño de San Justo; Argentina Fil: Alconcher, Laura. Provincia de Buenos Aires. Hospital Interzonal General Dr. José Penna; Argentina Fil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Amaral, María Marta. Universidad de Buenos Aires; Argentina. Universidad de Buenos Aires; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Hemolytic uremic syndrome (HUS), a vascular disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure, is caused by enterohemorrhagic Shiga toxin (Stx)-producing bacteria, which mainly affect children. Besides Stx, the inflammatory response mediated by neutrophils (PMN) is essential to HUS evolution. PMN can release neutrophil extracellular traps (NET) composed of DNA, histones, and other proteins. Since NET are involved in infectious and inflammatory diseases, the aim of this work was to investigate the contribution of NET to HUS. Plasma from HUS patients contained increased levels of circulating free-DNA and nucleosomes in comparison to plasma from healthy children. Neutrophils from HUS patients exhibited a greater capacity to undergo spontaneous NETosis. NET activated human glomerular endothelial cells, stimulating secretion of the proinflammatory cytokines IL-6 and IL-8. Stx induced PMN activation as judged by its ability to trigger reactive oxygen species production, increase CD11b and CD66b expression, and induce NETosis in PMN from healthy donors. During HUS, NET can contribute to the inflammatory response and thrombosis in the microvasculature and thus to renal failure. Intervention strategies to inhibit inflammatory mechanisms mediated by PMN, such as NETosis, could have a potential therapeutic impact towards amelioration of the severity of HUS. Fil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Sabbione, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Santiago, Adriana Patricia. Provincia de Buenos Aires. Ministerio de Salud. Hospital Municipal del Niño; Argentina Fil: Amaral, María Marta. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Exeni, Ramon. Provincia de Buenos Aires. Ministerio de Salud. Hospital Municipal del Niño; Argentina Fil: Trevani, Analía Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.

Haemolytic uraemic syndrome (HUS) is the major complication of Escherichia coli gastrointestinal infections that are Shiga toxin (Stx) producing. Monocytes contribute to HUS evolution by producing cytokines that sensitize endothelial cells to Stx action and migration to the injured kidney. As CC chemokine receptors (CCRs) are involved in monocyte recruitment to injured tissue, we analysed the contribution of these receptors to the pathogenesis of HUS. We analysed CCR1, CCR2 and CCR5 expression in peripheral monocytes from HUS patients during the acute period, with healthy children as controls. We observed an increased expression of CCRs per cell in monocytes from HUS patients, accompanied by an increase in the absolute number of monocytes CCR1+, CCR2+ and CCR5+. It is interesting that prospective analysis confirmed that CCR1 expression positively correlated with HUS severity. The evaluation of chemokine levels in plasma showed that regulated on activation of normal T-cell-expressed and -secreted (RANTES) protein was reduced in plasma from patients with severe HUS, and this decrease correlated with thrombocytopenia. Finally, the expression of the higher CCRs was accompanied by a loss of functionality which could be due to a mechanism for desensitization to compensate for altered receptor expression. The increase in CCR expression correlates with HUS severity, suggesting that the dysregulation of these receptors might contribute to an increased risk of renal damage. Activated monocytes could be recruited by chemokines and then receptors could be dysregulated. The dysregulation of CCRs and their ligands observed during the acute period suggests that a chemokine pathway would participate in HUS development. Fil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ruggieri, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Panek, Cecilia Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Exeni, Andrea. Hospital Municipal del Niño; Argentina Fil: Barilari, Catalina. Municipio de La Matanza. Hospital Municipal del Niño; Argentina Fil: Exeni, Ramon. Municipio de La Matanza. Hospital Municipal del Niño; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life. Fil: Mejias, Maria Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Cabrera, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Baschkier, Ariela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Ghersi, Giselle. Inmunova S.a; Argentina Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Miliwebsky, Elyzabeth. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Meiss, Roberto. Academia Nacional de Medicina. Centro de Estudios Oncológicos. Departamento de Patología; Argentina Fil: Goldbaum, Fernando Alberto. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Inmunova S.a; Argentina Fil: Zylberman, Vanesa. Inmunova S.a; Argentina. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina

Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx 2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx 2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer´s patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare. Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Cabrera, Gabriel Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Baschkier, Ariela. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Panek, Cecilia Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Miliwebsky, Elizabeth. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bentancor, Leticia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Rivas, Marta. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

The striking feature of Enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome (HUS). Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is presently available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure non toxicity but also a strong input to the immune system to induce long-lasting, high affinity antibodies with anti-Stx neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity for displaying foreign antigens on it. Taking into account BLS advantages and the potential capacity of the B subunit of Stx2 (Stx2B) to induce antibodies that prevent Stx2 toxicity by blocking its entrance to the host cells, we engineered a new immunogen inserting Stx2B at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce long-lasting humoral immune response in mice. The chimera induced antibodies with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2-challenge and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or antibody development with preventive or therapeutic ends, for use in HUS endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli. Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ghersi, Giselle. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Panek, Cecilia Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bentancor, Leticia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Baschkier, Ariela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Goldbaum, Fernando Alberto. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Zylberman, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Inmunova S.A; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo Inst. de Patobiología Fil: Martorelli, Luisina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Garbaccio, Sergio Gabriel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Vilte, Daniel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Albanese, Adriana Andrea. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos; Argentina Fil: Mercado, Elsa Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Ibarra, Cristina E. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS Fil: Bentancor, Leticia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bilen, Marcos Fabian. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Panek, Cecilia Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández, Gabriela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Isturiz, Martín Amadeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina