Development of an ADP-Ribosylation Assay for residual toxicity in C. difficile Binary Toxin CDTa using Automated Capillary Western Blot

Publication date: Available online 23 January 2020Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Richard R. Rustandi, Melissa HammAbstractCDTa, an actin ADP-ribosylation transferase, is a binary toxin produced by the bacterium Clostridium difficile which is commonly associated with the hypervirulent strain present in Clostridium difficile infections. The mutated form of CDTa, 4mCDTa, is one of the components in the tetravalent Clostridium difficile vaccine in which the residual toxicity of the ADP-ribosylation activity needs to be monitored for safety reasons. There are several ADP- ribosylation activity methods employing techniques such as ELISA, manual Western blot, or SDS PAGE, but all these methods are usually time consuming and labor intensive. Here we describe the development of new quantitative capillary based western for monitoring the presence of ADP-ribosylation activity in CDTa and 4mCDTa using novel, automated Simple Western™ technology. Furthermore, we have measured for the first time the enzyme’s kinetic parameters, KM (NAD) and kcat for native CDTa using this new quantitative capillary western technology.
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research

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Source: TIME: Health - Category: Consumer Health News Authors: Tags: Uncategorized 2019-nCoV Infectious Disease Source Type: news
Abstract Toxoid vaccines against Clostridium difficile infections (CDI) appear promising in reducing the risk of developing toxin-mediated symptoms. We sought to evaluate the effectiveness and cost-effectiveness of a vaccine candidate in a hospital setting. We developed an agent-based simulation model of nosocomial CDI in a 300-bed hospital. Targeting high-risk patients for vaccination, we estimated the reduction of symptomatic CDI. Using the net reduction of CDI-associated isolation days, we evaluated the vaccine's cost-effectiveness from a healthcare provider perspective over a 2-year period with an average mont...
Source: Vaccine - Category: Allergy & Immunology Authors: Tags: Vaccine Source Type: research
wska-Mach J Abstract Clostridioides (formerly Clostridium) difficile infections (CDIs) are becoming more common and more serious. C. difficile is the etiologic agent of antibiotic-associated diarrhea, pseudomembranous enterocolitis, and toxic megacolon while CDIs recur in 7.9% of patients. About 42.9 CDI cases/10,000 patient-days are diagnosed each day in Europe, whereas in Poland 5.6 CDI cases/10,000 patient-days are reported; however, the median for European countries is 2.9 CDI cases/10,000 patient-days. Epidemiology of CDIs has changed in recent years and risk of developing the disease has doubled in the past ...
Source: Acta Microbiologica et Immunologica Hungarica - Category: Microbiology Authors: Tags: Acta Microbiol Immunol Hung Source Type: research
Abstract Clostridioides difficile infection is the leading cause of nosocomial diarrhoea globally. Immune responses to toxins produced by C. difficile are important in disease progression and outcome. Here, we analysed the anti-toxin A and anti-toxin B serum antibody proteomes following natural infection or vaccination with a C. difficile toxoid A/toxoid B vaccine using a modified miniaturised proteomic approach based on de novo mass spectrometric sequencing. Analysis of immunoglobulin variable region (IgV) subfamily expression in immunoprecipitated toxin A and toxin B antibodies from four and seven participants o...
Source: Vaccine - Category: Allergy & Immunology Authors: Tags: Vaccine Source Type: research
Clostridioides difficile is the primary cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease resulting in a significant fatality rate. Colonization of the gut is critical for C. difficile pathogenesis. The bacterial molecules essential for efficient colonization therefore offer great potential as vaccine candidates. Here we present findings demonstrating that the C. difficile immunogenic lipoprotein CD0873 plays a critical role in pathogen success in vivo. We found that in a dixenic colonization model, a CD0873-positive strain of C. difficile significantly outcompeted a CD0873-neg...
Source: Journal of Biological Chemistry - Category: Chemistry Authors: Tags: Protein Structure and Folding Source Type: research
Purpose of review Clostridioides difficile infection (CDI) is a significant burden on the health system, especially due to high recurrence rates. Since the beginning of the CDI epidemic in early 2000s, many strategies for combatting recurrence have been explored, with moderate success so far. This review will focus on the most recent developments in recurrent CDI prevention and treatment. Recent findings There are two main mechanisms of CDI recurrence: alteration in microbiome and poor antibody response. Development of new antibiotics aims to minimize damage to the microbiome. Fecal transplant or other microbiome repl...
Source: Current Opinion in Infectious Diseases - Category: Infectious Diseases Tags: GASTROINTESTINAL INFECTIONS: Edited by Gagandeep Kang and Eric R. Houpt Source Type: research
Clostridium difficile disease is mediated primarily by toxins A and B (TcdA and TcdB, respectively). The receptor binding domains (RBD) of TcdA and TcdB are immunogenic, and anti-RBD antibodies are protective. Since these toxins act locally, an optimal C. difficile vaccine would generate both systemic and mucosal responses. We have repurposed an attenuated Salmonella enterica serovar Typhimurium strain (YS1646) to produce such a vaccine. Plasmid-based candidates expressing either the TcdA or TcdB RBD were screened. Different vaccine routes and schedules were tested to achieve detectable serum and mucosal antibody titers in...
Source: Infection and Immunity - Category: Infectious Diseases Authors: Tags: Microbial Immunity and Vaccines Source Type: research
Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous...
Source: Infection and Immunity - Category: Infectious Diseases Authors: Tags: Molecular Pathogenesis Source Type: research
Condition:   Clostridium Infections Interventions:   Biological: C. difficile investigational vaccine based on the F2 antigen (GSK2904545A);   Biological: C. difficile investigational vaccine based on the F2 antigen (GSK2904545A) adjuvanted with AS01B;   Drug: Placebo Sponsor:   GlaxoSmithKline Not yet recruiting
Source: ClinicalTrials.gov - Category: Research Source Type: clinical trials
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