Flexneri 2a Research Articles

Impact of O-Acetylation on S. flexneri 1b and 2a O-Antigen Immunogenicity in Mice.

Shigellosis is a diarrheal disease caused prevalently by Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines.

Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

Assessment of the Use of Gentian Violet and Malachite Green as Selective Agents in the Isolation of Brucella

The aim of the work is to evaluate ability of gentian violet and malachite green to inhibit 2 Brucella test strains and 8 strains of unwanted organisms and the influence of autoclaving on their activity. The study also aims to evaluate variability of inhibition in different batches of colorants and necessity in adjustment of their concentration.Materials and methods. The study included 3 imported lots of gentian violet and malachite green. Inhibitors were put into nutrient medium containing pancreatic hydrolysates of gelatin and fsh meal, yeast extract, sodium chloride, glucose, sodium pyrosulfate, and agar. The same medium without colorants was used as a control medium. Effect of the colorants was studied with the help of test strains of Brucella abortus 19 ВА, B. melitensis Rev I, Enterococcus faecium VKM B-602, Escherichia coli ATCC 25922, Proteus vulgaris HX 19 222, Pseudomonas aeruginosa 22/99, Serracia plymuthica 1, Shigella flexneri 1a 8516, Staphylococcus aureus ATCC 25923, Yersinia enterocolitica C-187.Results and discussion. We have determined biological parameters of nutrient media containing gentian violet (5,0; 2,5 1,25 mg/L) and malachite green (2.0; 1.0; 0.5 mg/L) when cultivating brucella. Gentian violet is able to inhibit Brucella greatly at concentration of 5 mg/L. S. aureus ATCC 25923 did not show any growth on the media containing malachite green or gential violet (5.0 and 2.5 mg/L). Malachite green inhibited the growth of enterococcus more actively than gentian violet. However, it inhibited swarming growth of Proteus less successfully than gentian violet. Other unwanted organisms were not sensitive to such concentrations of colorants at all. We have demonstrated the necessity to combine colorants with other selective agents, as well as advisability to put gentian violet into sterile media, while malachite green – into the media before its autoclaving. Adjustments to the concentration of gentian violet should be made when producing nutrient medium, as variations in inhibiting activity in different batches of this colorant were revealed.

Rationalizing the design of a broad coverage Shigella vaccine based on evaluation of immunological cross-reactivity among S. flexneri serotypes.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.

PSXVI-14 Study of the antagonistic properties of C. utilis in relation to the resident microflora of the animal organism

Abstract C. utilis yeast is widely used in industry for the production of fodder protein, amino acids, vitamins, and enzymes. We have studied the antimicrobial activity of C. utilis CY 302 against such collection strains as E. coli 3912/41, S. flexneri 1a 8516, S. enterica serovar Typhimurium 79, S. epidermidis ATCC 14990, S. aureus ATCC 25923, Proteus vulgaris HX 19 222, L. casei subsp. Rhamnosus ATCC 7469, B. breve ATCC 15701, C. albicans ATCC 10231 by using the agar blocks method and by measuring the inhibition growth zone (IGZ, mm) accompanied by the target cells death zone. The yeast strain was grown on Sabouraud’s medium at 28±1 °C for 3 days, and the test cultures were grown on differential diagnostic media for each bacterial species (HiMedia Laboratories Pvt. Ltd, India) at 35±2 °C overnight. The analyses were done separately for each culture. A weak antimicrobial effect was observed with respect to L. casei and B. breve, the IGZ was 5.0 and 4.0 mm, respectively. The halos of inhibition against pathogenic bacteria were 7 mm for E. coli, 5 mm for S. typhimurium, 5 mm for P. vulgaris, 6 mm for S. fiexneri, 6 mm for S. aureus, and 5 mm for S. epidermidis. After incubation there was noted the absence of DZZR in the wild C. utilis CY 302 strain in relation to the C. albicans ATCC 10231 strain from the collection. As a result of the studies the antagonistic potential of the yeast isolate C. utilis CY 302 was revealed. The work was supported by the Ministry of Science and Higher Education of Russia.

Profiling of Indigenous Biosurfactant-Producing Bacillus Isolates in the Bioremediation of Soil Contaminated by Petroleum Products and Olive Oil.

Petroleum is, up to this date, an inimitable nonrenewable energy resource. Petroleum leakage, which arises during transport, storage, and refining, is the most important contaminant in the environment, as it produces harm to the surrounding ecosystem. Bioremediation is an efficient method used to treat petroleum hydrocarbon-contaminated soil using indigenous microorganisms. The degradation characteristics for a variety of hydrocarbons (hexane, benzene, gasoline, and diesel) were qualitatively and quantitatively investigated using Bacillus isolates. Microbiological and biochemical methods have been used including isolation of oil-degrading bacteria, enzymatic activities, the determination of physicochemical parameters, biosurfactant production and extraction assay, oil displacement assay, antimicrobial assay of the biosurfactants, and bioremediation kinetics. Consequently, of the 60 isolates capable of degrading different hydrocarbons at fast rates, 34 were suspected to be Bacillus isolates capable of growing in 24 h or 48 h on BH medium supplemented with 2% of hexane, benzene, gasoline, diesel, and olive oil, respectively. Among the 34 isolates, 61% (21/34) are capable of producing biosurfactant-like molecules by using gasoline, 70% (24/34) with diesel oil, 85% (29/34) with hexane, and 82% (28/34) with benzene. It was found that biosurfactant-producing isolates are extractable with HCl (100%), ammonium sulphate (95%), chloroform (95%), and ethanol (100%). Biosurfactants showed stability at 20°C, 37°C, 40°C, and 60°C. Biosurfactant secreted by Bacillus strains has shown an antagonistic effect in Escherichia coli, Shigella flexneri 5a M90T, and Bacillus cereus. The selected isolates could therefore be safely used for biodegradation. Substrate biodegradation patterns by individual isolates were found to significantly differ. The study shows that benzene was degraded faster, followed by hexane, gasoline, and finally diesel. The Bacillus consortium used can decrease hydrocarbon content from 195 to 112 (g/kg) in 15 days.

Orthogonal modular biosynthesis of nanoscale conjugate vaccines for vaccination against infection.

Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases. Although nanotechnology has been greatly applied in the field of vaccines, it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers. In this work, an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system. When SpyTag/SpyCatcher system is combined with protein glycosylation technology, bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles (VLPs) in a biocompatible and controlled manner. After confirming the excellent lymph node targeting and humoral immune activation abilities, these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model. These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines. To the best of the researchers’ knowledge, this is the first time VLP-based nanoconjugate vaccines are produced efficiently, and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.Electronic Supplementary MaterialSupplementary material (Figs. S1–S9) is available in the online version of this article at 10.1007/s12274-021-3713-4.

Shigella-Specific Immune Profiles Induced after Parenteral Immunization or Oral Challenge with Either Shigella flexneri 2a or Shigella sonnei.

ABSTRACTShigella spp. are a leading cause of diarrhea-associated global morbidity and mortality. Development and widespread implementation of an efficacious vaccine remain the best option to reduce Shigella-specific morbidity. Unfortunately, the lack of a well-defined correlate of protection for shigellosis continues to hinder vaccine development efforts. Shigella controlled human infection models (CHIM) are often used in the early stages of vaccine development to provide preliminary estimates of vaccine efficacy; however, CHIMs also provide the opportunity to conduct in-depth immune response characterizations pre- and postvaccination or pre- and postinfection. In the current study, principal-component analyses were used to examine immune response data from two recent Shigella CHIMs in order to characterize immune response profiles associated with parenteral immunization, oral challenge with Shigella flexneri 2a, or oral challenge with Shigella sonnei. Although parenteral immunization induced an immune profile characterized by robust systemic antibody responses, it also included mucosal responses. Interestingly, oral challenge with S. flexneri 2a induced a distinctively different profile compared to S. sonnei, characterized by a relatively balanced systemic and mucosal response. In contrast, S. sonnei induced robust increases in mucosal antibodies with no differences in systemic responses across shigellosis outcomes postchallenge. Furthermore, S. flexneri 2a challenge induced significantly higher levels of intestinal inflammation compared to S. sonnei, suggesting that both serotypes may also differ in how they trigger induction and activation of innate immunity. These findings could have important implications for Shigella vaccine development as protective immune mechanisms may differ across Shigella serotypes.IMPORTANCE Although immune correlates of protection have yet to be defined for shigellosis, prior studies have demonstrated that Shigella infection provides protection against reinfection in a serotype-specific manner. Therefore, it is likely that subjects with moderate to severe disease post-oral challenge would be protected from a homologous rechallenge, and investigating immune responses in these subjects may help identify immune markers associated with the development of protective immunity. This is the first study to describe distinct innate and adaptive immune profiles post-oral challenge with two different Shigella serotypes. Analyses conducted here provide essential insights into the potential of different immune mechanisms required to elicit protective immunity, depending on the Shigella serotype. Such differences could have significant impacts on vaccine design and development within the Shigella field and should be further investigated across multiple Shigella serotypes.

Glycoconjugation of Shigella flexneri type 2a O-polysaccharide with CRM197 as a potential vaccine candidate for shigellosis

Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin. We derivatized CRM197 and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM197-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM197 ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis.

Shigella-specific antibodies in the first year of life among Zambian infants: A longitudinal cohort study.

IntroductionShigellosis, is a leading cause of moderate-to-severe diarrhoea and related mortality in young children in low and middle income countries (LMICs). Knowledge on naturally acquired immunity can support the development of Shigella candidate vaccines mostly needed in LMICs. We aimed to quantify Shigella-specific antibodies of maternal origin and those naturally acquired in Zambian infants.MethodsPlasma samples collected from infants at age 6, 14 and 52-weeks were tested for Shigella (S. sonnei and S. flexneri 2a) lipopolysaccharide (LPS) antigen specific immunoglobulin G (IgG) and A (IgA) by enzyme-linked immunosorbent assay.ResultsAt 6 weeks infant age, the IgG geometric mean titres (GMT) against S. sonnei (N = 159) and S. flexneri 2a (N = 135) LPS were 311 (95% CI 259–372) and 446 (95% CI 343–580) respectively. By 14 weeks, a decline in IgG GMT was observed for both S. sonnei to 104 (95% CI 88–124), and S. flexneri 2a to 183 (95% CI 147–230). Both S. sonnei and S. flexneri 2a specific IgG GMT continued to decrease by 52 weeks infant age when compared to 6 weeks. In 27% and 8% of infants a significant rise in titre (4 fold and greater) against S. flexneri 2a and S. sonnei LPS, respectively, was detected between the ages of 14 and 52 weeks.IgA levels against both species LPS were very low at 6 and 14 weeks and raised significantly against S. flexneri 2a and S. sonnei LPS in 29% and 10% of the infants, respectively.ConclusionIn our setting, transplacental IgG anti-Shigella LPS is present at high levels in early infancy, and begins to decrease by age 14 weeks. Our results are consistent with early exposure to Shigella and indicate naturally acquired IgG and IgA antibodies to S. flexneri 2a and S. sonnei LPS in part of infants between 14 and 52 weeks of age. These results suggest that a potential timing of vaccination would be after 14 and before 52 weeks of age to ensure early infant protection against shigellosis.

Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlate of protection

BackgroundShigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis. MethodsIn this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed. FindingsSixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002). InterpretationThe Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) FundingFunding for this study was through a grant from the Wellcome Trust.

Immune response characterization in a human challenge study with a Shigella flexneri 2a bioconjugate vaccine

BackgroundDiarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. MethodsFlexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. FindingsFlexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. InterpretationCollectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. FundingFunding for this study was provided through a Wellcome Trust grant.

Molecular Identification and Antimicrobial Sensitivity Profile of Bacteria Isolated from Food Handlers in Akure Nigeria

Hand washing and hand swab samples culminating to a total of 477 samples were collected from different food vending locations in Akure. Bacteria were isolated and identified, after which the sensitivity patterns of the isolates to standard antibiotics and plant extracts were determined using disc diffusion agar and well diffusion methods, respectively. The bacterial isolates include Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcus luteus, Proteus mirabilis, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella enteritica, Salmonella typhi, Serratia marcescens, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus aureus and, Streptococcus pyogenes. Staphylococcus aureus was observed to be the most prominent bacteria harboured by the food handlers. The isolated bacteria were subjected to 16s rRNA polymerase chain reaction, DNA gene extraction and sequence blasting using the amplified length of 1500 base pair. The sequence obtained was blasted in National Centre for Biotechnology Information (NCBI) database. Based on the 16s rRNA sequences, the following bacteria were confirmed; Escherichia coli str. K12 substr. DH10B, Klebsiella pneumoniae strain J1, Proteus mirabilis strain HI4320, Pseudomonas fluorescens SBW25, Salmonella enterica subsp.enterica serovar Typhi str. CT18, Salmonella enterica subsp. enterica serovar Infantis, Shigella flexneri 2a str. 2457T and Staphylococcus cohnii subsp. cohnii strain 532 Contig16. Phylogeny was employed in the phylogenetic tree construction due to its robustness, reliability and less-laboriousness. The result of the sequence was aligned and the phylogenetic tree constructed shows the evolutionary relationship between eight bacterial species. Two clusters were generated; Salmonella enterica subsp. enterica serovar Typhi, Salmoenella typhi LN854584.1 Staphylococcus cohnii, and Staphylococcus aureus are in cluster 1, Proteusmirabilis NC0100554.1, Bacillus thuringiensis KY652120.1 and Bacillus subtilis MG231261.1 is incluster 2. However, bacteria in the same cluster were observed to have different phenotypic characteristics. The antibacterial efficacy of the selected medicinal plant extracts (Ocimum gratissimum and Annona muricata) were copared to that of standard antibiotics (Ciprofloxacin, Nitrofurantoin, Ampliclox, and Chloramphenicol) using three different solvents (methanol, water and petroleum ether) at 50 mg/ml, 100 mg/ml, 200mg/ml and 300mg/ml, concentrations. Bioactive characterization of the selected plants revealed the presence ofvarious active components such as Thymol, Eugenol, Caryophyllene, Gamma-terpinene, 8-methyl-4-vanillyl-7-nonenamide, N-4-Hydroxy-3-methoxybenyl 8methyldec 6 enamide, DL-6, 8-tio acidamide and N-4-Hydroxy-3-methoxyphenyl methyl nonanamide. Phytochemical analysis of the plants affirmed the presence of Tannins, Alkaloids, Flavonoids, Anthraquinone and Saponins in O. gratissimum and A. muricata. It can be deduced from this study that food handlers play a major role in the transfer of bacterial contaminants to the edible food. Also, the metabolites present in O. gratissimum and A. muricata render these plants cost effective alternative source of therapy to food borne illnesses.

Redirecting substrate regioselectivity using engineered \u0394N123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

A retrospective analysis of Shigellosis in hospitalized patients

The relevance of the study of Shigellosis is due to the fact that the pathogen is capable of invasion and the production of cyto- and neurotoxins, which leads to the development of severe inflammation and toxicosis, and with massive infection — and neurotoxicosis. Purpose: to study the clinical and laboratory features of the course of Shigellosis in hospitalized patients at the present stage. Materials and Methods: a retrospective study of 17 case histories of children aged 9 month to 17 years old, hospitalized in the infectious diseases department of a Moscow hospital in 2019 with a confirmed diagnosis of Shigellosis, was carried out. Results: depending on the season, the frequency of hospitalizations of children with Shigellosis was practically the same in autumn, winter and spring, excluding summer (35%, 35%, 30% and 0%, respectively). Children with Shigellosis under the age of 1 year were 2, 1—3 years old — 4, 3—7 years old — 5, 7—14 years old — 3, over 14 years old — 3. The diagnosis of Shigellosis was confirmed in 13 patients according to the results of bacteriological research , in 4 — according to IHR (indirect hemagglutination reaction) data (without seeding). Shigella flexneri 2a was detected in 14 children, Shigella sonnei in 2, Shigella flexneri 2a together with Shigella sonnei in 1. Most of the patients 82.4% (14/17) were admitted to the hospital in the first three days of illness (by 2.8 ± 0.05 days). Intoxication symptoms were expressed in all patients, vomiting — in 76.5% of cases (13/17), fever — in 94% (16/17), abdominal pain — in 82.4% (14/17), inflammatory impurities in feces (cloudy mucus) — in all patients, hemocolitis (streaks of blood in feces) — in all but one — 94% (16/17). In one third of patients, Shigellosis was severe — in 35% (6/17). Conclusions. Shigellosis remains relevant due to the incidence of hemocolitis, severe forms, which requires hospitalization.

Public health and clinical implications of multidrug-resistant shigellosis cases in a reception centre for refugees/migrants, Greece, October-December 2019.

From October to December 2019, 18 shigellosis cases (median age: 4.3 years, range: 0-21) were identified in the Reception Center for refugees/migrants on the Greek island of Samos. Fifteen cases (83.3%) were Afghani. Median time from arrival to symptoms onset was 53 days (40-101). Isolates from 14 cases, serotyped as Shigella flexneri 1b, and from three cases, serotyped as S. sonnei phase S (I), presented a multidrug-resistant phenotype. S. flexneri 1b isolates also produced extended-spectrum β-lactamases. Shigella flexneri 2a isolate from the remaining case was resistant to sulfomethoxazole, trimethoprim and pefloxacin. Improvement of hygiene and strengthening of laboratory investigation is needed.

Zinc deficiency induces epithelial barrier dysfunction and altered intestinal ion transport in novel murine model of Shigella flexneri diarrhea

Background: Zinc (Zn) deficiency has been linked with etiology for wide range of gastrointestinal diseases. Although low serum Zn has been associated with diarrhea and growth retardation, however the underlying mechanism of Zn deficiency aggravating the diarrheal manifestation remains elusive. Here, we have studied the physiological cascade elicited during Zn deficiency in both colonic cell lines and murine model leading to altered intestinal barrier function and ion transport. Methods and materials: T84 were grown onto transwell inserts in Zn deficient conditions for measurements of transepithelial electrical resistance (TER) and short-circuit current (Isc). Adult BALB/c mice fed with Zn deficient diet for 14 days and were intraperitoneally challenged with virulent S.flexneri 2a for following assessments of electrophysiological properties of the colonized colon in ussing chamber. Colonization evaluation from feces and colon homogenate were further analyzed for bacterial translocation assay. Results: Zn deficient cells infected with Shigella flexneri 2a showed significant increase in transmigration of the pathogens (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) for 6 h post infection along with poor formation of transepithelial resistance (1014 + 165 Ωcm2 vs 3663 + 293 Ωcm2). Ultrastructure of T84 cells grown in Zn deficient medium showed widened TJ complex and due to few membrane fusion proteins showing distorted TJ morphology and shorthened microvilli. Immunoblotting analysis further showed increased expression of Claudin-2 leaky tight junction protein in Zn deficient cells by modulating ERK dependent pathways. Following studies with Ussing chambers further demonstrated increase in Ca2+ dependent electrogenic Cl− secretion in Zn deficient cells. In vivo studies showed Zn deficient condition in mice increased bacterial translocation, colonization, bacterial shedding and pronounced disruption in intestinal permeability. Conclusion: Our results suggest that in T84 cells and murine of Shigellosis, Zn deficiency causes (1) pronounced barrier dysfunction and (2) altered Cl− secretion leading to heightened diarrheal pathogenesis thus linking micronutrient deficiency with bacterial infection and inflammation.

Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells.

Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen.IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study

Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 μg (cohort 1) and 10 μg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 μg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 μg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 μg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. The European Union Seventh Framework Programme.

Immunization With Outer Membrane Vesicles Derived From Major Outer Membrane Protein-Deficient Salmonella Typhimurium Mutants for Cross Protection Against Salmonella Enteritidis and Avian Pathogenic Escherichia coli O78 Infection in Chickens.

Colibacillosis is an economically important infectious disease in poultry, caused by avian pathogenic Escherichia coli (APEC). Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne diseases in human circulated through poultry-derived products, including meat and chicken eggs. Vaccine control is the mainstream approach for combating these infections, but it is difficult to create a vaccine for the broad-spectrum protection of poultry due to multiple serotypes of these pathogens. Our previous studies have shown that outer membrane vesicles (OMVs) derived from S. enterica serovar Typhimurium mutants with a remodeled outer membrane could induce cross-protection against heteroserotypic Salmonella infection. Therefore, in this study, we further evaluated the potential of broad-spectrum vaccines based on major outer membrane protein (OMP)-deficient OMVs, including ΔompA, ΔompC, and ΔompD, and determined the protection effectiveness of these candidate vaccines in murine and chicken infection models. The results showed that ΔompA led to an increase in the production of OMVs. Notably, ΔompAΔompCΔompD OMVs showed significantly better cross-protection against S. enterica serovar Choleraesuis, S. Enteritidis, APEC O78, and Shigella flexneri 2a than did other omp-deficient OMVs, with the exception of ΔompA OMVs. Subsequently, we verified the results in the chicken model, in which ΔompAΔompCΔompD OMVs elicited significant cross-protection against S. Enteritidis and APEC O78 infections. These findings further confirmed the feasibility of improving the immunogenicity of OMVs by remodeling the outer membrane and provide a new perspective for the development of broad-spectrum vaccines based on OMVs.