scholarly journals Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

2015 ◽  
Vol 84 (2) ◽  
pp. 395-406 ◽  
Author(s):  
Kasper Krogh Andersen ◽  
Nika M. Strokappe ◽  
Anna Hultberg ◽  
Kai Truusalu ◽  
Imbi Smidt ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Neutralizing Antibody ◽  
Treated Group ◽  
Western World ◽  
Hamster Model ◽  
Toxin B ◽  
Content Type ◽  
Vhh Antibodies ◽  
Clostridium Difficile Toxin B

Clostridium difficileis the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors ofC. difficileare two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineeredLactobacillusstrains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdBin vitrowere isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form inLactobacillus paracaseiBL23, they were able to neutralize the cytotoxic effect of the toxin in anin vitrocell-based assay. Prophylactic treatment with a combination of two strains of engineeredL. paracaseiBL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA−TcdB+strain ofC. difficile(P< 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized withC. difficilefor up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

scholarly journals Memory B Cells Encode Neutralizing Antibody Specific for Toxin B from the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 but with Superior Neutralization of VPI 10463 Toxin B

2015 ◽  
Vol 84 (1) ◽  
pp. 194-204 ◽  
Author(s):  
T. Scott Devera ◽  
Gillian A. Lang ◽  
Jordi M. Lanis ◽  
Pragya Rampuria ◽  
Casey L. Gilmore ◽  
...  
Keyword(s):  
B Cells ◽  
Clostridium Difficile ◽  
Mononuclear Cells ◽  
Neutralizing Antibody ◽  
Memory B Cells ◽  
Lethal Challenge ◽  
Toxin B ◽  
Content Type ◽  
Antibody Titers

Secreted toxin B (TcdB) substantially contributes to the pathology observed duringClostridium difficileinfection. To be successfully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encoding memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vaccination against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD) fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI 10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 induced strong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferred to naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Further experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible with Bmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells (PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven antibody titers, and the CTD2 antibody was neutralizingin vitro, but its protection against lethal challenge with TcdB2 was limited to delaying time to death. Therefore, CTD from differentC. difficilestrains may be a good immunogen for stimulating B cell memory that encodesin vitroneutralizing Ab but may be limited by variable protection against intoxicationin vivo.

scholarly journals A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

2015 ◽  
Vol 22 (7) ◽  
pp. 711-725 ◽  
Author(s):  
Natalie G. Anosova ◽  
Leah E. Cole ◽  
Lu Li ◽  
Jinrong Zhang ◽  
Anna M. Brown ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Toxin A ◽  
Healthy Human ◽  
Hamster Model ◽  
Toxin B ◽  
Content Type ◽  
Highly Virulent

ABSTRACTClostridium difficileinfection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and Bin vitroand because of theirin vivoefficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains ofC. difficiletoxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

scholarly journals MBX-500, a Hybrid Antibiotic withIn VitroandIn VivoEfficacy against Toxigenic Clostridium difficile

2012 ◽  
Vol 56 (9) ◽  
pp. 4786-4792 ◽  
Author(s):  
Michelle M. Butler ◽  
Dean L. Shinabarger ◽  
Diane M. Citron ◽  
Ciarán P. Kelly ◽  
Sofya Dvoskin ◽  
...  
Keyword(s):  
Weight Gain ◽  
Clostridium Difficile ◽  
Severe Disease ◽  
Normal Weight ◽  
New Drugs ◽  
Hamster Model ◽  
Resistance Development ◽  
Content Type

ABSTRACTClostridium difficileinfection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, thein vitroandin vivoefficacies of MBX-500 were explored against the Gram-positive anaerobe,C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

scholarly journals In VitroandIn VivoActivities of the Novel Ketolide RBx 14255 against Clostridium difficile

2012 ◽  
Vol 56 (11) ◽  
pp. 5986-5989 ◽  
Author(s):  
Manoj Kumar ◽  
Tarun Mathur ◽  
Tarani K. Barman ◽  
G. Ramkumar ◽  
Ashish Bhati ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Time Dependent ◽  
Kinetics Study ◽  
The Novel ◽  
Promising Candidate ◽  
Hamster Model ◽  
Bacterial Killing ◽  
Content Type ◽  
Time Kill

ABSTRACTThe MIC90of RBx 14255, a novel ketolide, againstClostridium difficilewas 4 μg/ml (MIC range, 0.125 to 8 μg/ml), and this drug was found to be more potent than comparator drugs. Anin vitrotime-kill kinetics study of RBx 14255 showed time-dependent bacterial killing forC. difficile. Furthermore, in the hamster model ofC. difficileinfection, RBx 14255 demonstrated greater efficacy than metronidazole and vancomycin, making it a promising candidate forC. difficiletreatment.

scholarly journals The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4222-4232 ◽  
Author(s):  
Dennis Bakker ◽  
Anthony M. Buckley ◽  
Anne de Jong ◽  
Vincent J. C. van Winden ◽  
Joost P. A. Verhoeks ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Virulence Factors ◽  
Pathogenic Bacteria ◽  
Microarray Data Analysis ◽  
Toxin A ◽  
Hamster Model ◽  
Golden Syrian Hamster ◽  
Content Type

ABSTRACTIn the past decade,Clostridium difficilehas emerged as an important gut pathogen. Symptoms ofC. difficileinfection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that theC. difficile630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its rolein vivoandin vitrothrough the creation of an isogenic ClosTron-basedhtrAmutant ofC. difficilestrain 630Δerm(wild type). In contrast to the attenuated phenotype seen withhtrAdeletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acuteC. difficileinfection. Microarray data analysis showed a pleiotropic effect ofhtrAon the transcriptome ofC. difficile, including upregulation of the toxin A gene. In addition,the htrAmutant showed reduced spore formation and adherence to colonic cells. Together, our data show thathtrAcan modulate virulence inC. difficile.

scholarly journals Bacteriophage Combinations Significantly Reduce Clostridium difficile GrowthIn Vitroand ProliferationIn Vivo

2015 ◽  
Vol 60 (2) ◽  
pp. 968-981 ◽  
Author(s):  
Janet Y. Nale ◽  
Janice Spencer ◽  
Katherine R. Hargreaves ◽  
Anthony M. Buckley ◽  
Przemysław Trzepiński ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Oral Delivery ◽  
Therapeutic Potential ◽  
Bacterial Load ◽  
Range Analysis ◽  
Hamster Model ◽  
Content Type ◽  
Phage Types ◽  
Specific Phage

ABSTRACTThe microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse ofClostridium difficileinfection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplificationin situ, but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of sevenC. difficilephages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis ofC. difficilein vitroand prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reducedC. difficilecolonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI.

scholarly journals Human Monoclonal Antibodies Directed against Toxins A and B Prevent Clostridium difficile-Induced Mortality in Hamsters

2006 ◽  
Vol 74 (11) ◽  
pp. 6339-6347 ◽  
Author(s):  
Gregory J. Babcock ◽  
Teresa J. Broering ◽  
Hector J. Hernandez ◽  
Robert B. Mandell ◽  
Katherine Donahue ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Human Monoclonal Antibodies ◽  
Toxin A ◽  
Hamster Model ◽  
Cytotoxic Effects ◽  
Toxin B ◽  
Reduction Of Mortality

ABSTRACT Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P < 0.0001) in the primary disease hamster model and from 78% to 32% (P < 0.0001) in the less stringent relapse model.

scholarly journals The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

2016 ◽  
Vol 84 (12) ◽  
pp. 3434-3444 ◽  
Author(s):  
Kevin O. Childress ◽  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Sarah E. Anderson ◽  
Emily C. Woods ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Clostridium Difficile ◽  
Molecular Mechanisms ◽  
Toxin Production ◽  
Negative Regulator ◽  
Hamster Model ◽  
Content Type ◽  
Sporulation Initiation

The formation of spores is critical for the survival ofClostridium difficileoutside the host gastrointestinal tract. Persistence ofC. difficilespores greatly contributes to the spread ofC. difficileinfection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation toC. difficilepathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved inC. difficileand few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion ofCD1492resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in theCD1492mutant. Deletion of CD1492 also resulted in decreased toxin productionin vitroand in decreased virulence in the hamster model of CDI. Further, theCD1492mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lowersigDandrstAtranscription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence thatC. difficilesporulation is regulated differently from that of other endospore-forming species.

scholarly journals A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels ofIn VitroPotency ShowsIn VivoProtection in a Hamster Infection Model

2013 ◽  
Vol 20 (3) ◽  
pp. 377-390 ◽  
Author(s):  
Nicola L. Davies ◽  
Joanne E. Compson ◽  
Brendon MacKenzie ◽  
Victoria L. O'Dowd ◽  
Amanda K. F. Oxbrow ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Neutralizing Antibodies ◽  
Disease Process ◽  
Care Facility ◽  
Infection Model ◽  
Hamster Model ◽  
Content Type ◽  
Toxin Binding

ABSTRACTClostridium difficileinfections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments,C. difficileinfection (CDI) is still a major cause of patient suffering, death, and substantial health care costs.Clostridium difficileexerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety ofin vitrobinding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

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