Mucosal IFNγ production and potential role in protection in Escherichia coli O157:H7 vaccinated and challenged cattle.

Shiga-toxin producing Escherichia coli O157:H7 (O157)-based vaccines can provide a potential intervention strategy to limit foodborne zoonotic transmission of O157. While the peripheral antibody response to O157 vaccination has been characterized, O157-specific cellular immunity at the rectoanal junction (RAJ), a preferred site for O157 colonization, remains poorly described. Vaccine induced mucosal O157-specific antibodies likely provide some protection, cellular immune responses at the RAJ may also play a role in protection. Distinct lymphoid follicles were increased in the RAJ of vaccinated/challenged animals. Additionally, increased numbers of interferon (IFN)γ-producing cells and γδ + T cells were detected in the follicular region of the RAJ of vaccinated/challenged animals. Likewise, adjuvanted-vaccine formulation is critical in immunogenicity of the O157 parenteral vaccine. Local T cell produced IFNγ may impact epithelial cells, subsequently limiting O157 adherence, which was demonstrated using in vitro attachment assays with bovine epithelial cells. Thus, distinct immune changes induced at the mucosa of vaccinated and challenged animals provide insight of mechanisms associated with limiting O157 fecal shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling O157 in ruminants and thus limiting O157 transmission to humans.

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.

Identification of Nanobodies Blocking Intimate Adherence of Shiga Toxin-Producing Escherichia coli to Epithelial Cells.

Therapeutic antibodies (Abs) inhibiting bacterial adhesion to host epithelia are an attractive option to reduce the load of Shiga toxin-producing E. coli (STEC) in the intestine of the patient and also in the bovine reservoir, thereby minimizing the risk of STEC contamination in the food chain. Of particular interest are recombinant single-domain Ab fragments called nanobodies (Nbs) derived from the variable domain of camelid heavy chain-only antibodies (VHH). The outer membrane adhesin intimin and the translocated intimin receptor (Tir) are essential for the attachment of STEC to host epithelia. In addition, EspA filaments of the bacterial type III protein secretion system are needed for Tir translocation into the host cell. Given their importance for bacterial adhesion and colonization, we developed Nbs against intimin, Tir and EspA proteins of STEC serotype O157:H7. Here, we report the screening methods used to isolate inhibitory Nbs blocking intimin-Tir protein-protein interaction, actin-pedestal formation, and intimate adhesion of STEC to epithelial cells in vitro. First, we describe how VHH gene repertoires can be produced as Nbs secreted by E. coli using the α-hemolysin (HlyA) protein secretion system. Next, we report the methods for identification of inhibitors of intimin-Tir protein-protein interaction and of STEC intimate adhesion to HeLa cells in culture. These methods can be adapted for the screening of Nbs against different adhesin-receptor complexes to block the adhesion of other pathogens to host cells.

Differential Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.

Microfluidics for the rapid detection of Escherichia coli O157:H7 using antibody-coated microspheres.

This study developed a novel method for the rapid detection of Escherichia coli (E. coli) O157:H7 on a microfluidic platform. First, the concentration of bacteria in a sample was determined with the adenosine triphosphate (ATP) method. Then, the specific detection of E. coli was achieved in a microfluidic chip by the immune-microsphere technique. The influences of the culture time, flow rate and capture time on the detection of the target bacteria were investigated systematically. Generally, with increasing capture time, more bacteria could be captured by the microspheres, which had a positive effect on bacterial detection. Furthermore, the sensitivity and specificity of the method were also tested. The results showed that this method could specifically detect E. coli with a sensitivity as high as 49.1 cfu/µL; the consumption of bacteria was 1 µL, and the reagent was at the microliter level. The testing time can be controlled within one and a half hours, and the cost of testing was approximately RMB 10. The method described in this article is simple and accurate and has great application value in bacterial detection for medical diagnostics.

Identification of novel monoclonal antibodies targeting the outer membrane protein C and lipopolysaccharides for Escherichia coli O157:H7 detection.

AIMS: To identify and evaluate the application of two novel monoclonal antibody (mAb) 2G12 against outer membrane protein (Omp) C and mAb 12B1 targeting the O chain of the lipopolysaccharides (LPS) of Escherichia coli O157:H7 (ECO157). METHODS AND RESULTS: The sensitivity and specificity of these two antibodies were evaluated with eight ECO157 strains and 68 untargeted strains. mAb 2G12 and 12B1 had no detectable binding with any of the non-O157 strains at 6·0 log10 CFU per ml, while its high specificity and affinity remained with all ECO157 strains. When a higher level (8·0 log10 CFU per ml) was tested, 2G12 and 12B1 did not react with 82·35 and 97·06% of the non-O157 strains respectively. Based on the pair of two antibodies, the sandwich enzyme-linked immunosorbent assay detected 100% (8/8) of ECO157 strains and none of the non-ECO157 strains. The detection limit of ECO157 strains in pure culture were 4·2 ± 0·2 log10 CFU per ml. When the developed test was applied to artificially inoculated beef samples, the detection limit was 6·0 log10 CFU per gram without enrichment and 1·0 log10 CFU per gram after 12 h of enrichment. CONCLUSIONS: The two novel antibodies identified in this study served as great candidates for the recovery, and detection of ECO157 from different environmental and food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: ECO157-specific detection was improved by a combination of the novel OmpC mAb and LPS mAb with defined target antigen and good specificity.

Synthesis of PDA-Mediated Magnetic Bimetallic Nanozyme and Its Application in Immunochromatographic Assay.

Immunochromatographic assay (ICA) is widely applied in various fields. However, severe matrix interference and weak signal output present major challenges in achieving accurate and ultrasensitive detection in ICA. Here, a polydopamine (PDA)-mediated magnetic bimetallic nanozyme (Fe3O4@PDA@Pd/Pt) with peroxidase-like activity was synthesized and used as a probe in ICA. The magnetic property of Fe3O4@PDA@Pd/Pt enabled effective magnetic enrichment of targets, thereby reducing the matrix interference in the sample. PDA coating on the magnetic bimetallic nanozyme was employed as a mediator and a stabilizer. It improved the catalytic ability and stability of the magnetic bimetallic nanozyme by providing more coordination sites for Pd/Pt growth and functional groups (-NH and -OH). In addition, the Pd/Pt bimetallic synergistic effect could further enhance the catalytic ability of the nanozyme. A method was developed by integrating Fe3O4, PDA, and Pd/Pt into Fe3O4@PDA@Pd/Pt as a probe in ICA. With the proposed method, human chorionic gonadotropin and Escherichia coli O157:H7 were successfully detected to be as low as 0.0094 mIU/mL in human blood serum and 9 × 101 CFU/mL in the milk sample, respectively. This method may be readily adapted for accurate and ultrasensitive detection of other biomolecules in various fields.

Repeated Oral Vaccination of Cattle with Shiga Toxin-Negative Escherichia coli O157:H7 Reduces Carriage of Wild-Type E. coli O157:H7 after Challenge.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE+E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 109 CFU of a pool of three stx-negative LEE+E. coli O157:H7 strains (vaccine group) or three stx-negative LEE- non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx+ LEE+E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ.IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Immunoassay for foodborne pathogenic bacteria using magnetic composites Ab@Fe3O4, signal composites Ap@PtNp, and thermometer readings.

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe3O4 were incubated in Cu2+ phosphate buffer to synthesize the magnetic composite Ab@Fe3O4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu2+ phosphate buffer to synthesize the signal composites Ap@PtNp with antimicrobial peptides (magainin I), recognizing and labeling E. coli O157:H7. In the presence of E. coli O157:H7, magnetic microcomposites targeted bacteria and signal microcomposites to form the sandwich structure: Ab@Fe3O4-bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H2O2 to generate thermo-signals (temperature rise), which were determined by a thermometer. This point-of-care bioassay detected E. coli O157:H7 in the linear range of 101-107 CFU mL-1 and with a detection limit of 14 CFU mL-1. One-pot process magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4, magnetic microcomposites, MMC) for immunomagnetic separation and nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp, signal microcomposites, SMC) were used as signal amplification and thermometer readings for E. coli O157:H7 detection.

A colorimetric immunoassay for determination of Escherichia coli O157:H7 based on oxidase-like activity of cobalt-based zeolitic imidazolate framework.

Cobalt-based zeolitic imidazolate framework nanosheets (ZIF-67) with oxidase-like catalytic activities as an immunoprobe were employed to enhance the sensitivity of an immunoassay. ZIF-67 was synthesized via the solvothermal method using 2-methylimidazole and cobalt dichloride as substrates. A colorimetric immunoassay for Escherichia coli (E. coli) O157:H7 was designed. Preparation of the immunoprobe involved self-polymerized dopamine being applied for the surface modification of ZIF-67 nanosheets in order to bind to the antibody, which was used to identify E. coli O157:H7. ZIF-67 catalyze the oxidation of 3,3',5,5'-tetramethylbiphenyl (TMB) and produced a color change from colorless to blue. Upon reaction termination, the absorbance was measured at 450 nm. By combining ZIF-67@PDA catalyzed chromogenic reaction with antibody recognition and magnetic separation, the limit of determination is 12 CFU mL-1 and the linear range is 30 to 3.0 × 108 CFU mL-1. The proposed colorimetric immunoassay was successfully utilized to detect E. coli O157:H7 of spiked food samples. Graphical abstract.

Fe3O4@CuS-based immunochromatographic test strips and their application to label-free and dual-readout detection of Escherichia coli O157:H7 in food.

Herein, a label-free and dual-readout immunochromatographic test strip (ITS) for the sensitive detection of Escherichia coli (E. coli) O157:H7 by taking advantages of the strong capture ability of Fe3O4@CuS nanostructures (NSs) towards bacteria and their ultrahigh photothermal effects (PTEs) was reported. Especially, without the customarily antibody (Ab)-labeled probe, Fe3O4@CuS NSs could be adsorbed onto the surfaces of bacteria to form Fe3O4@CuS-bacteria conjugates and then trapped by immobilized Abs on the test line (T-line), forming a characteristic yellow band. After direct immunoreactions, the PTEs of Fe3O4@CuS NSs endowed T-line to be irradiated by an 808 nm infrared light, obtaining satisfactory sensitivity and accuracy. Under optimal conditions, E. coli O157:H7, as low as 103 and 102 CFU/mL, could be monitored in colorimetric and photothermal modes. Additionally, E. coli O157:H7 was successfully detected in beef, chicken, milk and honey samples by this proposed platform with a recovery of 80-120%.

Functional nanozyme mediated multi-readout and label-free lateral flow immunoassay for rapid detection of Escherichia coli O157:H7.

To overcome the drawbacks of antibody labeling dependence and single-readout system in the conventional lateral flow immunoassays (LFIAs) as well as the non-targeted combination of new capture agents reported recently for pathogen detection, in this work, a multi-readout and label-free LFIA was proposed for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) based on a nanozyme-bacteria-antibody sandwich pattern. A type of functional nanozyme-mannose modified Prussian blue (man-PB), was introduced as the recognition agent as well as signal indicator. Apart from original signal intensity on the T-line, the peroxidase-like catalytic activity-driven generation of colorimetric signal could be used as another format of quantitation. Importantly, such LFIA could exhibit excellent performance for target pathogens detection separately with a quantitative range of 102-108 cfu·mL-1 and a low detection limit of 102 cfu·mL-1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples.

Heterologous expression of Intimin and IpaB fusion protein in Lactococcus lactis and its mucosal delivery elicit protection against pathogenicity of Escherichia coli O157 and Shigella flexneri in a murine model.

Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group.In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens.

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection.

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 103 CFU per ml and for H7 positive E. coli is 1 × 104 CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 104 CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.

Cattle intestinal microbiota shifts following Escherichia coli O157:H7 vaccination and colonization

Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p <0.05) association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p < 0.05). The Firmicutes:Bacteroidetes ratio (p > 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7.

Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function.

Lipocalin 2 (Lcn2) is an essential component of the antimicrobial innate immune system. It attenuates bacterial growth by binding and sequestering the iron-scavenging siderophores to prevent bacterial iron acquisition. Whereas, the ability of Lcn2 to sequester iron is well-described, the role of Lcn2 in regulating immune cells during bacterial infection remains unclear. In this study, we showed that upon infection with Escherichia coli (O157:H7), Lcn2-deficient (Lcn2-/-) mice carried more bacteria in blood and liver, and the acute-phase sera lost their antibacterial activity in vitro. Neutrophils from Lcn2-/- mice were defective in homeostasis and morphological development. E. coli O157:H7 infection of Lcn2-/- mice resulted in a reduced neutrophil migration capacity, with 30% reduction of extravasated neutrophils, and impaired chemotaxis, as shown by a reduction in the secretion of chemoattractants, such as tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2, which are instrumental in eliciting a neutrophil response. We also found that some secreted cytokines [interleukin (IL)-6, IL-1ß, and TNF-α] were decreased. Transcripts of inflammatory cytokines (IL-6, IL-1ß, TNF-α, and IL-10), chemokines (MIP-2 and MCP-1), and iNOS production were all strongly repressed in Lcn2-/- macrophages. Furthermore, Lcn2 could induce the production of chemokines and promote the migration and phagocytosis of macrophages. Thus, Lcn2 deficiency could impair the migration and chemotaxis ability of neutrophils and disturb the normal secretion of inflammatory cytokines of macrophages. Therefore, the heightened sensitivity of Lcn2-/- mice to E. coli O157:H7 is not only due to the antibacterial function of Lcn2 but also a consequence of impaired functions of immune cells, including neutrophils and macrophages.

Identification of lipid A deacylase as a novel, highly conserved and protective antigen against enterohemorrhagic Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is a major cause of large outbreaks worldwide associated with hemorrhagic colitis and hemolytic uremic syndrome. While vaccine development is warranted, a licensed vaccine, specific for human use, against EHEC is not yet available. In this study, the reverse vaccinology approach combined with genomic, transcriptional and molecular epidemiology data was applied on the EHEC O157:H7 genome to select new potential vaccine candidates. Twenty-four potential protein antigens were identified and one of them (MC001) was successfully expressed onto Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing this vaccine candidate was immunogenic, raising a specific antibody response. Immunization with the MC001 candidate was able to reduce the bacterial load of EHEC O157:H7 strain in feces, colon and caecum tissues after murine infection. MC001 is homologue to lipid A deacylase enzyme (LpxR), and to our knowledge, this is the first study describing it as a potential vaccine candidate. Gene distribution and sequence variability analysis showed that MC001 is present and conserved in EHEC and in enteropathogenic E. coli (EPEC) strains. Given the high genetic variability among and within E. coli pathotypes, the identification of such conserved antigen suggests that its inclusion in a vaccine might represent a solution against major intestinal pathogenic strains.

Entrapment of HETS recombinant protein onto PLGA and alginate NPs improves the immunogenicity of the protein against E. coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) are known as the gastrointestinal pathogens and major causes of enterohemorrhagic colitis since decades ago. There is no efficient approved vaccine against EHEC O157 and non-O157. In the present study, a recombinant candidate vaccine against enterohemorrhagic E. coli (EHEC) O157:H7 entrapped in the sodium alginate and PLGA nanoparticles and the efficiency of the immunization of these formulations were investigated. nanoparticles due to their properties like controlled cargoes release, adjuvanticity, cargo protection, increased bioavailability, etc have been noticed for drug delivery. A chimeric protein composed of HcpA, EspA, Tir and Stx2B antigens was designed, recombinantly expressed, purified and entrapped in nanoparticles. BALB/c mice were administrated with nano-formulated and free proteins. IgG titer, EHEC fecal shedding and the ability of the immune sera to neutralize Stx toxin and inhibit the bacterial attachment to Caco-2 cells were analyzed. Fecal shedding analysis demonstrated that the colonization of the bacteria in the intestine of the mice was reduced significantly (P > 0.01). Immune mice were able to tolerate up to 200 LD50 of the active Stx toxin. About 80% of the bacterial binding capacity to Caco-2 cells was declined, especially in groups immunized with nano-formulations. Considering the importance of EHEC, especially O157 serotype, on public health and the other hand, the lack of an efficient vaccine in this regard, delivery of HETS candidate vaccine with NPs can be applied to prevent the infection by the pathogen.

Development of a Gold Nanoparticle Vaccine against Enterohemorrhagic Escherichia coli O157:H7.

Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichiacoli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens.IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models.

Oral immunization of mice with Lactococcus lactis expressing Shiga toxin truncate confers enhanced protection against Shiga toxins of Escherichia coli O157:H7 and Shigella dysenteriae.

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin-inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL-Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD50 of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL-Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae.