Development and clinical validation of an automated cell cytotoxicity neutralization assay for detecting Clostridioides difficile toxins in clinically relevant stools samples.

OBJECTIVES: To improve the diagnostic accuracy of Clostridioides difficile infection, current U.S. and E.U. guidelines recommend multistep testing that detects the presence of C. difficile and toxin in clinically relevant stool samples to confirm active disease. An accepted gold standard to detect C. difficile toxins is the cell cytotoxicity neutralization assay (CCNA). Although highly sensitive, the traditional CCNA has limitations. One such limitation is the subjective interpretation of an analyst to recognize cytopathic effects in cultured cells exposed to a fecal sample containing toxin. To overcome this limitation, an automated CCNA was developed that replaces most human pipetting steps with robotics and incorporates CellTiterGlo® for a semi-quantitative, non-subjective measure of cell viability instead of microscopy. METHODS: To determine sample positivity and control for non-specific cytopathic effects, two thresholds were defined and validated by evaluating the sample with/without antitoxin antisera (sample-antitoxin/sample + antitoxin): 1) a >70% cell viability threshold was validated with samples containing anti-toxin, and 2) a >1.2-fold difference cut-off where sample results above the cut-off are considered positive. RESULTS: Assay validation demonstrated excellent accuracy, precision, and sample linearity with an LOD of 126.9 pg/mL toxin-B in stool. The positivity cut-offs were clinically validated by comparing 322 diarrheal stool sample results with those run in a predicate, microscopic readout-based CCNA. The automated CCNA demonstrated 96% sensitivity and 100% specificity compared with the predicate CCNA. CONCLUSIONS: Overall, the automated CCNA provides a specific, sensitive, and reproducible tool to support determination of CDI epidemiology or the efficacy of interventions such as vaccines.

Type VII Toxin/Antitoxin Classification System for Antitoxins that Enzymatically Neutralize Toxins.

Toxin/antitoxin (TA) systems are present in nearly all bacterial and archaeal strains and consist of a toxin that reduces growth and an antitoxin that masks toxin activity. Currently there are six primary classes for TA systems based on the nature of the antitoxin and the way that the antitoxin inactivates the toxin. Here we show that there now are at least three additional and distinct TA systems in which the antitoxin is an enzyme and the cognate toxin is the direct target of the antitoxin: Hha/TomB (antitoxin oxidizes Cys18 of the toxin), TglT/TakA (antitoxin phosphorylates Ser78 of the toxin), and HepT/MntA (antitoxin adds three AMPs to Tyr104 of the toxin). Thus, we suggest the type VII TA system should be used to designate those TA systems in which the enzyme antitoxin chemically modifies the toxin post-translationally to neutralize it. Defining the type VII TA system using this specific criterion will aid researchers in classifying newly discovered TA systems as well as refine the framework for recognizing the diverse biochemical functions in TA systems.

Proposed reduction of the in vivo pyrogen test by the in vitro LAL assay for the quality control of anticrotallic, antiscorpion, antirabies and antitetanus sera.

The rationale for a formal study of the LAL (kinetic chromogenic method) assay for anticrotallic (SAC), antirabies (SAR), antitetanus (SAT) and antiscorpion (SAE) sera involved the determination of parameters required by the Brazilian National Health Surveillance Agency (ANVISA), the USA Food and Drug Administration (FDA), USP (United States Pharmacopeia, 39) and ICH (International Conference on Harmonization). The curve correlation coefficients obtained with the standard endotoxin control ranged from -0.980 to -1.000 in all experiments. Endotoxin recovery added to the SAC, SAR, SAT and SAE samples, at the working dilutions (1:10, 1:10, 1:10 and 1, 100 respectively), met the criteria required by the FDA, USP and Brazilian ANVISA for the Inhibition-Potentiation test. The applied methodology for the four analyzed sera fulfilled the required criteria for all performance parameters. Thus, the present study demonstrated that the in vivo pyrogen test can be potentially replaced by the LAL assay for all assessed sera samples displaying higher sensitivity and following the 3 Rs principle, in addition to maintaining quality control in Sanitary Surveillance.

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.

The role of the humoral immune response to Clostridium difficile toxins A and B in susceptibility to C. difficile infection: a case-control study.

Antibody levels to Clostridium difficile toxin A (TcdA), but not toxin B (TcdB), have been found to determine risk of C. difficile infection (CDI). Historically, TcdA was thought to be the key virulence factor; however the importance of TcdB in disease is now established. We re-evaluated the role of antibodies to TcdA and TcdB in determining patient susceptibility to CDI in two separate patient cohorts. In contrast to earlier studies, we find that CDI patients have lower pre-existing IgA titres to TcdB, but not TcdA, when compared to control patients. Our findings suggest that mucosal immunity to TcdB may be important in the early stages of infection and identifies a possible target for preventing CDI progression.

Antioxidant, antimicrobial and antiverotoxic potentials of extracts of Curtisia dentata.

ETHNOPHARMACOLOGICAL RELEVANCE: There is an increase in antimicrobial resistance and complexities arising from verotoxic related bacterial infections as well as rise in demand for application of natural antioxidants to combat oxidative damage by free radicals in many oxidative stress-mediated disease conditions such as cancer. Thus the potential of Curtisia dentata as antimicrobial, antioxidant and antiverotoxin against environmental isolates of Escherichia coli and Acinetobacter spp. as well as the presence of phytochemicals and some organic compounds, was determined. MATERIALS AND METHODS: Phytochemical analysis was carried out using standard methods and antioxidant activity was determined using the DPPH radical scavenging activity. Effect of extracts on bacterial cell wall was also determined. RESULTS: Extracts contained anthraquinones, alkaloids, essential oils, glycosides, phenols, steroids, saponins, tannins, quinones, anthocyanins, amines and carboxylic acids as phytochemicals. Extracts demonstrated high antimicrobial activity and low minimum inhibitory concentrations as well as inhibitory action against the expression of both Vtx1 and Vtx2 genes in Escherichia coli, Acinetobacter haemolyticus and Acinetobacter lwoffii. Ethanol root bark extracts consistently showed the highest DPPH radical scavenging activity (62.43%), total phenol content (TPH) (57.62 26 mg GAE/g) and reducing power (RP) (41.32%), followed by those of the stem bark and leaf extracts with the respective values of 54.68%, 37.77 mg GAE/g and 21.83%. The extracts induced the leakage of Na(+) and K(+) ions from both test bacteria. CONCLUSION: Curtisia dentata is a very effective source of antioxidant and a possible alternative to sourcing antiverotoxic antibiotics with novel mechanism of action.

[The interaction between chromosome-encoded toxin Slr0664 and antitoxin Slr1114 of cyanobacteria Synechocystis sp. PCC6803].

OBJECTIVE: We identified the interaction between toxin Slr0664 and antitoxin Slr1114, encoded by ssr114/ slr0664 system in the chromosome of cyanobacteria Synechocystis sp. PCC6803. METHODS: We constructed a recombinant plasmid in which only H6-Ssr1114 was induced to express, and another plasmid in which both H6-Ssr1114 and Slr0664 was co-expressed in E. coli B121(DE3). After induction, we used affinity capture technique to purify H,-Ssr1114 and copurified H6-Ssr1114 and Slr0664 under different conditions. We confirmed the co-purified H,-Ssrl 114 and S1l0664 by using mass spectrographic analysis. RESULTS: When induced to express, Slr0664 showed cell toxicity leading to cell growth suppression or death. However, cells could grow normally if both H6-Ssr1114 and Slr0664 were induced to co-express. We could purify both H6-Ssr1114 and Slr0664 by His-Bind under native conditions, but only H4-Ssr1114 could be purified under denature conditions. The results of mass spectrometric analysis showed that the copurified proteins were H,-Ssrlll4 and Slr0664. CONCLUSION: The antitoxin Slr1114 and toxin Slr0664 in ssr1114/slr0664 TA system was interacted with each other.

Gaining ground: assays for therapeutics against botulinum neurotoxin.

Owing in part to recently heightened concern over bioterrorism, interest in the mechanism of action of botulinum neurotoxin (BoNT) and development of effective therapeutic strategies has dramatically increased. The emergence of BoNT as an effective treatment for a variety of neurological disorders and its growing use in the cosmetic industry have also increased interest in developing effective countermeasures. Although recent attempts to create effective vaccines appear promising, the multitude of clinical and cosmetic uses of BoNT make mass vaccination against the toxin undesirable and impractical, leading to intensified efforts to develop effective therapeutics to combat large-scale intoxications. In this review, we examine the relevant and available in vitro cell-based assays and in vivo assays for drug discovery and development, especially with regard to the potential for medium- to high-throughput automation and its use in identifying physiologically relevant inhibitors.

Quantification of potency of neutralizing antibodies to botulinum toxin using compound muscle action potential (CMAP).

We evaluated a method for quantifying botulinum toxin-neutralizing antibodies which utilizes the CMAP. This method can be used just one day after administration, and the detection sensitivity was higher than that of the mouse neutralization test. The CMAP neutralization test detected neutralizing antibodies in patients who were resistant to treatment with the botulinum LL toxin. These results indicate that the CMAP neutralization test is useful for detecting low levels of antitoxin.

Intramuscular delivery of a cholera DNA vaccine primes both systemic and mucosal protective antibody responses against cholera.

Cholera is a potentially lethal diarrhea disease caused by the gram-negative bacterium Vibrio cholerae. The need for an effective cholera vaccine is clearly indicated but the challenges of eliciting both systemic and mucosal immune responses remains a significant challenge. In the current report, we discovered that a DNA vaccine expressing a protective cholera antigen, cholera toxin B subunit (CTB), delivered parenterally can elicit both systemic and mucosal anti-CTB antibody responses in mice. The priming effect by DNA immunization was demonstrated by higher mucosal antibody responses following one boost with the inactivated cholera vaccine (KWC-B) delivered orally when compared to the twice oral administration of KWC-B alone. This finding indicates that DNA vaccines delivered parenterally are effective in eliciting mucosal protective immune responses--a unique advantage for DNA vaccination that has not yet been well realized and should bring value to the development of novel vaccination approaches against mucosally transmitted diseases.

Induction of high antitoxin titers against tetanus toxoid in rabbits by intranasal immunization with dextran microspheres.

Poor absorption of protein antigens through the mucosal membranes necessitates the use of mucoadhesive delivery systems. Regarding the advantages of mucosal immunization and also the penetration enhancement potential of dextran microspheres, in this study the adjuvant potential of these microspheres was compared with CpG-ODN. Cross-linked dextran microspheres (CDMs) were loaded with tetanus toxoid (TT). In vitro release studies were performed in a model, simulating the nasal cavity. The immunoreactivity of encapsulated TT was assayed by ELISA. Membrane toxicity and local irritating potential of CDM was examined by erythrocyte hemolysis and nasal administration to human nose, respectively. The various formulations were nasally administered to rabbits (n=4). Alum-adsorbed TT (AATT) was injected as the positive control. The serum IgG and nasal lavage sIgA titers were determined by ELISA method. Serum antitoxin titers were determined by toxin neutralization (TN) bioassay method. Mean diameter of CDM was 128.1+/-25.8 microm. Mean encapsulation efficiency was 20.3+/-3.2% (n=3). Antigenicity of encapsulated TT was 90.5+/-1.8% (n=3) that of original TT. Hemolysis studies showed no membrane disruption by CDM and none of the human subjects reported nasal irritation. Among the nasally immunized animals, the highest antitoxin titers was seen in the group immunized with CDM+TT (P<0.0001). The serum IgG titers of the CDM+TT group was higher than the TT solution group (P<0.05). The adjuvant potentials of CDM and CpG-ODN in inducing IgG titers was not significantly different (P>0.05). The lowest sIgA titers in the bronchial lavage were seen in the group of animals received AATT parenterally. Considering the proper release characteristics, desirable preservation of the antigen activity of TT, good mucoadhesion properties and also safety of CDM+TT, these microspheres could be regarded as an efficient mucosal adjuvant and antigen delivery system. These microspheres could induce very high antitoxin titers following nasal administration, while the CpG-ODN could not induce such titers. The antitoxin titers induced by CDM+TT was 175 times higher than the protective levels.

Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B.

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library.

BACKGROUND: Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. RESULTS: A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. CONCLUSION: We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles.

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.

Evidence for widespread epithelial damage and coincident production of monocyte chemotactic protein 1 in a murine model of intestinal ricin intoxication.

The development of small-animal models is necessary to understand host responses and immunity to emerging infectious diseases and potential bioterrorism agents. In this report we have characterized a murine model of intestinal ricin intoxication. Ricin administered intragastrically (i.g.) to BALB/c mice at doses ranging from 1 to 10 mg/kg of body weight induced dose-dependent morphological changes in the proximal small intestine (i.e., duodenum), including widespread villus atrophy and epithelial damage. Coincident with epithelial damage was a localized increase in monocyte chemotactic protein 1, a chemokine known to be associated with inflammation of the intestinal mucosa. Immunity to intestinal ricin intoxication was achieved by immunizing mice i.g. with ricin toxoid and correlated with elevated levels of antitoxin mucosal immunoglobulin A (IgA) and serum IgG antibodies. We expect that this model will serve as a valuable tool in identifying the inflammatory pathways and protective immune responses that are elicited in the intestinal mucosa following ricin exposure and will prove useful in the evaluation of antitoxin vaccines and therapeutics.

Qualification and performance characteristics of a quantitative enzyme-linked immunosorbent assay for human lgG antibodies to anthrax lethal factor antigen.

The contribution of Bacillus anthracis lethal factor (LF)-specific immune responses to protection against anthrax disease in humans remains incompletely defined due, in part, to a paucity of qualified reagents and a lack of standardized serological assays. Toward this end, we have identified and characterized suitable positive quality control and standard reference sera and developed, optimized, and qualified an enzyme-linked immunosorbent assay (ELISA) to measure LF-binding IgG. Herein we describe the performance characteristics of this ELISA and propose criteria for its use in the detection and quantification of anti-LF IgG in human serum.

Breast milk reduces the risk of illness in children of mothers with cholera: observations from an epidemic of cholera in Guinea-Bissau.

BACKGROUND: A protective effect of breastfeeding against cholera has been demonstrated in areas endemic of cholera. To assess the protection offered by breast milk from mothers living in an area that had been free from cholera for 7 years, we investigated mothers with cholera and their children during an epidemic with Vibrio cholerae El Tor in the capital of Guinea-Bissau. METHODS: Eighty mothers with clinical cholera and their children were identified, and interviewed. Blood samples for vibriocidal and antitoxin antibodies were collected from mother-and-child pairs. Breast milk samples were collected from lactating mothers. Cholera was defined as acute watery diarrhea during the epidemic and a vibriocidal reciprocal titer of 20 or above. RESULTS: Three (7%) of 42 breastfed children had cholera as defined above compared with 9 (24%) of 38 nonbreastfed children (RR for breastfed children, 0.19; 95% CI, 0.04-0.91, adjusted for age). The 3 breastfed children who developed cholera received milk containing lower concentrations of anticholera toxin IgA/total IgA (median, 2.0 units/mL) than 14 children who had serologic signs of colonization but did not develop the disease (median, 17.4 units/mL). CONCLUSIONS: The protective effect of breast milk against cholera is not confined to endemic areas. Lactating mothers with cholera should receive supportive care to continue breastfeeding.

Detection of anti-pertussis toxin IgG in oral fluids for use in diagnosis and surveillance of Bordetella pertussis infection in children and young adults.

Bordetella pertussis infection is being increasingly recognized as a cause of prolonged, distressing cough (without whooping symptoms) in children and young adults. Diagnosis of infection in this population is important for treatment and surveillance purposes, and may also prove useful in reducing transmission to unvaccinated babies, for whom disease can be fatal. Serum IgG titres against pertussis toxin (PT) are routinely used as a marker of recent or persisting B. pertussis infection. However, collection of serum from young children is difficult, and compliance amongst these subjects to give samples is low. To circumvent these problems, an IgG-capture ELISA capable of detecting anti-PT IgG in oral fluid was devised. The assay was evaluated by comparison to a serum ELISA, using 187 matched serum and oral fluid samples from children (aged 5-16 years) with a history of prolonged coughing, whose serum anti-PT titre had already been determined (69 seropositive, 118 seronegative). The results showed that, using a cutoff of 70 arbitrary units (AU), the oral fluid assay detected seropositive subjects with a sensitivity of 79.7% [95% confidence interval (CI) 68.3-88.4] and a specificity of 96.6% (95% CI 91.5-99.1). Thus, oral fluid titres of >or=70 AU would possess a positive predictive value of 76.2-93.2% for pertussis amongst children with chronic coughs when used as a surrogate for the serum ELISA (assuming disease prevalence of 12-37%). This oral fluid ELISA will greatly assist in the convenience of B. pertussis disease diagnosis and surveillance.

Pertussis antitoxin decay after vaccination with DTPa. Response to a first booster dose 3 1/2-6 1/2 years after the third vaccine dose.

Longitudinal serum samples were collected from 542 children that had participated in a Swedish pertussis vaccine trial 1992-1995 [Gustafsson L, Hallander HO, Olin P, Reizenstein E, Storsaeter J. A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med 1996;334(6):349-355] and who did not contract pertussis. The sera were analyzed for post vaccination antibody decay and for booster response of anti-PT (IgG antibodies against pertussis toxin), as measured by ELISA. Generally, an initial rapid decay of antitoxin antibody concentration was followed by a slower decay; the change occurring when the geometric mean level of antitoxin concentration reached 8-9 ELISA Units/mL (EU/mL). The time needed to reach this level was 8-9 months after the third dose in a 2, 4, and 6 months schedule. A "best-fit" combined regression model was used to predict when 50% of the children have less than the minimum level of detection of anti-PT (1EU/mL). This occurred about 65 months after dose 3 at an age of 6 years. The anti-PT response to a booster dose was evident but the post-booster geometric mean values decreased with number of years after the third dose and the response appeared later. The results indicate that a pre-school booster might be considered at 6 years of age or earlier.