An approach to chimeric subunit immunogen provides efficient protection against toxicity, type III and type v secretion systems of Shigella.

OBJECTIVES: Shigellosis is one of the infectious diseases causing severe intestinal illness in human beings. Development of an effective vaccine against Shigella is a key to deal with this bacterium. The present study aimed at evaluation of the antibody response as well as the protection of the recombinant chimeric protein containing IpaD, IpaB, StxB, and VirG against Shigella dysentery and flexneri. METHODS: Chimeric protein was expressed and purified by Ni-NTA resin. The identity of the protein was determined by Western blot analysis. Mouse groups were immunized with the recombinant protein and the humoral immune response was measured by Enzyme-Linked Immunosorbent Assay (ELISA). Additionally, neutralization of the bacterial toxin by antibody was assessed by MTT assay. Animal challenge against S.dysentery and S. flexneri was evaluated, as well. RESULTS: Protein expression and purification were confirmed by SDS-PAGE and western blotting. Analysis of the immune responses demonstrated that the antibody responses were higher in the sera of the subcutaneously immunized mice compared to those immunized intraperitoneally. In vitro neutralization analysis indicated that the 1:10000 dilution of the sera had a high ability to neutralize 0.25 ng/µl (CD50) of the toxin on the Vero cell line. Furthermore, the results of the animal challenge showed that the immunized mice were completely protected against 50 LD50 of the bacterial toxin. Immunization also protected 80% of the mice from 10 LD50 by S. flexneri and S.dysentery. In addition, passive immunization conferred 60% protection in the mice against S. flexneri and S.dysentery. Organ burden studies also revealed a significant reduction in infection among the immunized mice. CONCLUSION: This study revealed that the chimeric protein produced inE. colicould be a promising chimeric immunogen candidate against Shigella.

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections.

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents.

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA).

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

An in-vitro study on a novel six-phage cocktail against multi-drug resistant-ESBL Shigella in aquatic environment.

Shigella spp. are water-borne pathogens responsible for mild to severe cases bacilli dysentery all around the world known as Shigellosis. The progressively increasing of antibiotic resistance among Shigella calls for developing and establishing novel alternative therapeutic methods. The present study aimed to evaluate a novel phage cocktail of lytic phages against extended spectrum beta lactamase isolates of Shigella species in an aquatic environment. The phage cocktail containing six novel Shigella specific phages showed a broad host spectrum. The cocktail was very stable in aquatic environment. The cocktail resulted in about 99% decrease in the bacterial counts in the contaminated water by several species and strains of Shigella such as Shigella sonnei, Shigella flexneri and Shigella dysenteriae. Achieving such a high efficiency in this in-vitro study demonstrates a high potential for in-vivo and in-situ application of this phage cocktail as a bio-controlling agent against Shigella spp. contamination and infections.

In silico design of a vaccine candidate based on autotransporters and HSP against the causal agent of shigellosis, Shigella flexneri.

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.

Isolation, characterization and genomic analysis of a novel lytic bacteriophage vB_SsoS-ISF002 infecting Shigella sonnei and Shigella flexneri.

PURPOSE: Shigellosis is one of the most important food-borne and water-borne diseases worldwide. Although antibiotics are considered as efficient agents for shigellosis treatment, improper use of these has led to the emergence of antibiotic-resistant Shigella spp. Therefore, finding a new strategy as alternative treatment seems necessary. METHODOLOGY: Different samples from a wastewater treatment plant were used to isolate Shigella spp. specific phages. Physiological properties were determined, and genomic analysis was also carried out. RESULTS: A virulent Siphoviridae bacteriophage, vB_SsoS-ISF002, was isolated from urban wastewater in Iran and showed infectivity to different isolates of both Shigella sonnei and Shigella flexneri. vB_SsoS-ISF002 was stable at different pH values and temperatures. It had a short latent period (15 min), a large burst size (76±9 p.f.u. cell-1) and appropriate lytic activity especially at high MOI. Its genome (dsDNA) was 50 564 bp with 45.53 % GC content and 76 predicted open reading frames. According to comparative genomic analysis and phylogenic tree construction, vB_SsoS-ISF002 was considered as a member of the T1virus genus. CONCLUSION: These results indicated that vB_SsoS-ISF002 is a novel virulent T1virus phage and may have potential as an alternative treatment for shigellosis.

Shigellosis.

Shigellosis is a clinical syndrome caused by invasion of the epithelium lining the terminal ileum, colon, and rectum by Shigella species. Although infections occur globally, and in people of all ages, endemic infections among children aged 1-4 years living in low-income and middle-income settings constitute most of the disease burden. The versatile manifestations of these highly contagious organisms range from acute watery diarrhoea to fulminant dysentery characterised by frequent scant bloody stools with fever, prostration, and abdominal cramps. A broad array of uncommon, but often severe, intestinal and extraintestinal complications can occur. Despite marked reductions in mortality during the past three decades, there are roughly 164 000 annual deaths attributable to shigellosis. Intercontinental dissemination of multiresistant shigella strains, facilitated by travellers and men who have sex with men, has prompted new recommendations for antibiotic therapy. Awareness of disease burden and the emerging threats posed by shigella have accelerated interest in development of shigella vaccines, many of which are being tested in clinical trials.

[Hemolytic and uremic syndrome and related thrombotic microangiopathies: Treatment and prognosis]. / Syndromes hémolytiques et urémiques (SHU) et syndromes de microangiopathie thrombotique apparentés : traitement et pronostic.

Major achievements in the understanding of thrombotic microangiopathies (TMA) have not only resulted in a reclassification of TMA but most of all they have culminated in the design of new treatments and have enabled clinicians to better delineate their prognosis. Recent multicenter studies have improved our understanding of the prognosis of atypical hemolytic and uremic syndromes (aHUS). More specifically, they have highlighted the role of genetic testing on predicting the recurrence of aHUS, the risk of chronic kidney disease and the recurrence following kidney transplantation. A major advance consisted of the identification of the alternative complement pathway in the pathogenesis of aHUS, thus paving the way for the use of the C5a inhibitor eculizumab in this indication. Eculizumab has thereafter dramatically improved the management of patients affected with aHUS. During spring 2011, a great epidemic of entero-hemorrhagic Escherichia coli (EHEC) associated HUS occurred in Germany, providing clinicians the opportunity to examine the relevance of antibiotic prophylaxis, plasma exchange and eculizumab in EHEC-associated HUS. In this work, we herein present advances achieved in the setting of therapeutic management and prognosis in HUS and other related TMA syndromes.

A Synthetic Carbohydrate Conjugate Vaccine Candidate against Shigellosis: Improved Bioconjugation and Impact of Alum on Immunogenicity.

Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.

Management of acute infectious diarrhea for children living in resource-limited settings.

Acute infectious gastroenteritis continues to be a leading cause of morbidity and mortality in children below 5 years of age, with the majority of deaths concentrated in 35 'low income' countries. In these countries the under five years of age mortality rates reach 100 per 1000 live births, of which a significant proportion are associated with acute diarrhea. Rotavirus, cryptosporidium, Shigella spp and enterotoxigenic Escherichia coli are the main pathogens causing disease in these settings, although other bacteria and parasites can cause moderate to severe disease in different regions and situations. Treatment of children in these setting should be focused on appropriate rehydration, early hospitalization of severely malnourished children, zinc supplementation, and in specific situations, antimicrobials should be considered. The rationale for antimicrobial use should be based on the potential benefits based on published literature and the opportunity for use. This review provides a pathogen-specific update on the potential benefits of antimicrobials and suggests an empirical management approach for children suffering an acute watery or bloody diarrhea in a resource-limited region.

Hyperimmune bovine colostrum for treatment of GI infections: a review and update on Clostridium difficile.

Hyperimmune bovine colostrum (HBC), produced by vaccination of a cow during gestation, is rich in targeted immunoglobulins, and can be used to treat a variety of diseases. The published history of HBC use for treating gastrointestinal infections in humans has developed over the past several decades and demonstrates the promise of this type of therapeutic for GI infectious disease. HBC, or purified derivative products, have been used successfully for treatment or prevention of cryptosporidiosis, shigellosis, rotavirus, enterotoxigenic E. coli, and C. difficile infection (CDI). Given the positive results of previous studies using HBC for treatment of CDI, we have produced HBC with antibodies against the two most important virulence factors of C. difficile, TcdA and TcdB, using a novel recombinant vaccine. Our preliminary results demonstrate efficacy of the HBC product for treatment of CDI in the gnotobiotic piglet model, and warrant more thorough investigation. HBC may provide an effective treatment alternative to antibiotics, which can spare the normal gut microflora, and reduce rates of recurrence and antibiotic resistance.

Two live attenuated Shigella flexneri 2a strains WRSf2G12 and WRSf2G15: a new combination of gene deletions for 2nd generation live attenuated vaccine candidates.

Shigella infections are a major cause of inflammatory diarrhea and dysentery worldwide. First-generation virG-based live attenuated Shigella strains have been successfully tested in phase I and II clinical trials and are a leading approach for Shigella vaccine development. Additional gene deletions in senA, senB and msbB2 have been engineered into second-generation virG-based Shigella flexneri 2a strains producing WRSf2G12 and WRSf2G15. Both strains harbor a unique combination of gene deletions designed to increase the safety of live Shigella vaccines. WRSf2G12 and WRSf2G15 are genetically stable and highly attenuated in both cell culture and animal models of infection. Ocular immunization of guinea pigs with either strain induces robust systemic and mucosal immune responses that protect against homologous challenge with wild-type Shigella. The data support further evaluation of the second-generation strains in a phase I clinical trial.

Epidemiological study of shigellosis in an urban area of Argentina.

INTRODUCTION: Shigellosis represents one of the main causes of bloody diarrhoea in South America. This study aimed to establish the incidence of shigellosis in an urban zone of Buenos Aires, Argentina, by examining the type of Shigella and living conditions associated with this infection. METHODOLOGY: Between January 2009 and December 2010 we analyzed shigellosis in children admitted to the public health service with bloody diarrhoea from La Plata, the capital of Buenos Aires, Argentina. A total of 372 children under 15 years old with Shigella present in their stool samples were admitted to the study. Variables studied were patient age, type of Shigella, family economic status, and access to sewage services and safe drinking water. RESULTS: Shigella flexneri was found to be present in 66.8% of the cases. Incidence was 187 cases/year/100,000 children under 15 years old. Cases were mainly observed during the summer (38.5%) in the population of under 5 years old (69.1% of all cases). The risk of shigellosis increased 12 times in those children who lacked safe drinking water and this risk increased 1.5 times in the population without sewage services. Fewer cases of shigellosis were noted in downtown areas, while hot spots were identified in the suburbs. Treating one case of shigellosis has a local cost of US $976 while assuring safe drinking water and sewage services for one family costs US $634.  CONCLUSION: Incidence of shigellosis in urban areas is associated with quality of water and sewage services. Policies aimed at providing education and improving public utilities networks can help to reduce the incidence of shigellosis.

Natural products modulate Shigella-host-cell interaction.

This study focused on identifying possible new options derived from natural sources for the treatment of bacterial infections. Several natural products were investigated for their potential in modulating Shigella-host-cell interactions. The proliferation of Shigella sonnei was effectively inhibited inside HEp-2 cells in the presence of 4-methoxycinnamic acid and propolin D. Propolin D also significantly reduced the apoptosis of infected macrophage-like U937 cells and moderately reduced the secretion of interleukin (IL)-1ß and IL-18, which probably resulted from the inhibition of invasion plasmid antigen B secretion by this compound. Further characterization showed that propolin D did not prevent escape of Shigella from phagocytic vacuoles, as evidenced by actin-based motility and by the fact that addition of chloroquine did not further reduce the number of intracellular c.f.u. The role of propolin D in modulating autophagy could not be established under the experimental conditions used. As these compounds had no direct anti-Shigella activity in vitro, it was concluded that these compounds modulated Shigella-host-cell interactions by targeting yet-to-be defined mechanisms that provide benefits to host cells.

Green banana reduces clinical severity of childhood shigellosis: a double-blind, randomized, controlled clinical trial.

BACKGROUND AND AIMS: Mature green banana (GB) fruit is rich in amylase-resistant starch that stimulates colonic production of short-chain fatty acids (referred to as fatty acid) and is useful in treating diarrheal diseases. We studied therapeutic effects of GB in childhood shigellosis by determining colonic fatty acid production in a double-blind, randomized, controlled, clinical trial. METHODS: Seventy-three children aged 6 to 60 months with severe bloody dysentery caused by Shigella infection were either given a rice-based diet (54 kcal/dL), with cooked GB (250 g/L) (n = 34) or without GB (n = 39) for 5 days; all given ciprofloxacin (15 mg/kg, q12 hours). Stool volume, frequency, excretion of blood/mucus, and relevant clinical and laboratory indices were determined. RESULTS: On day 5 (post-treatment), 59% children in GB group had no mucus compared with 36% in controls, fecal blood was completely cleared from 96% in GB group compared with 60% without GB (P < 0.05). GB treatment significantly reduced (P < 0.01) numbers of stools/day compared with controls (70% vs. 50%, P < 0.05). GB-specific reductions of mean fecal volumes (mL/kg) ranged from 25% to 40%; (P < 0.05) during the 5-day observations. Clinical success rates were 85% in GB group compared with 67% in controls (P < 0.05). GB significantly (P < 0.01) reduced fecal myeloperoxidase activity and increased fecal fatty acid concentrations (P < 0.01). CONCLUSIONS: GB diet improves clinical severity in childhood shigellosis and could be a simple and useful adjunct for dietary management of this illness.