Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes

ABSTRACTClostridium difficileinfection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality.C. difficilemediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blockingC. difficiletoxin A- and toxin B-mediated inflammatory responses and histologic damage.

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Fidaxomicin and OP-1118 Inhibit Clostridium difficile Toxin A- and B-Mediated Inflammatory Responses via Inhibition of NF-κB Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01513-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 1

Author(s):

Hon Wai Koon ◽

Jiani Wang ◽

Caroline C. Mussatto ◽

Christina Ortiz ◽

Elaine C. Lee ◽

...

Keyword(s):

Clostridium Difficile ◽

Intestinal Inflammation ◽

Inflammatory Responses ◽

Human Colon ◽

Toxin A ◽

Primary Metabolite ◽

Necrosis Factor Alpha ◽

Toxin B ◽

Content Type ◽

Tnf Α

ABSTRACTClostridium difficilecauses diarrhea and colitis by releasing toxin A and toxin B. In the human colon, both toxins cause intestinal inflammation and stimulate tumor necrosis factor alpha (TNF-α) expression via the activation of NF-κB. It is well established that the macrolide antibiotic fidaxomicin is associated with reduced relapses ofC. difficileinfection. We showed that fidaxomicin and its primary metabolite OP-1118 significantly inhibited toxin A-mediated intestinal inflammation in micein vivoand toxin A-induced cell roundingin vitro. We aim to determine whether fidaxomicin and OP-1118 possess anti-inflammatory effects against toxin A and toxin B in the human colon and examine the mechanism of this response. We used fresh human colonic explants, NCM460 human colonic epithelial cells, and RAW264.7 mouse macrophages to study the mechanism of the activity of fidaxomicin and OP-1118 against toxin A- and B-mediated cytokine expression and apoptosis. Fidaxomicin and OP-1118 dose-dependently inhibited toxin A- and B-induced TNF-α and interleukin-1β (IL-1β) mRNA expression and histological damage in human colonic explants. Fidaxomicin and OP-1118 inhibited toxin A-mediated NF-κB phosphorylation in human and mouse intestinal mucosae. Fidaxomicin and OP-1118 also inhibited toxin A-mediated NF-κB phosphorylation and TNF-α expression in macrophages, which was reversed by the NF-κB activator phorbol myristate acetate (PMA). Fidaxomicin and OP-1118 prevented toxin A- and B-mediated apoptosis in NCM460 cells, which was reversed by the addition of PMA. PMA reversed the cytoprotective effect of fidaxomicin and OP-1118 in toxin-exposed human colonic explants. Fidaxomicin and OP-1118 inhibitC. difficiletoxin A- and B-mediated inflammatory responses, NF-κB phosphorylation, and tissue damage in the human colon.

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

Infection and Immunity ◽

10.1128/iai.00982-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6339-6347 ◽

Cited By ~ 168

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.

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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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00763-14 ◽

2015 ◽

Vol 22 (7) ◽

pp. 711-725 ◽

Cited By ~ 22

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.

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Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis

Journal of Clinical Microbiology ◽

10.1128/jcm.01810-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3702-3704 ◽

Cited By ~ 25

Author(s):

Grace O. Androga ◽

Julie Hart ◽

Niki F. Foster ◽

Adrian Charles ◽

David Forbes ◽

...

Keyword(s):

Ulcerative Colitis ◽

Clostridium Difficile ◽

Young Patient ◽

Diagnostic Methods ◽

Binary Toxin ◽

Toxin A ◽

Toxin B ◽

Content Type ◽

Severe Diarrhea ◽

Clinically Significant

Large clostridial toxin-negative, binary toxin-positive (A−B−CDT+) strains ofClostridium difficileare almost never associated with clinically significantC. difficileinfection (CDI), possibly because such strains are not detected by most diagnostic methods. We report the isolation of an A−B−CDT+ribotype 033 (RT033) strain ofC. difficilefrom a young patient with ulcerative colitis and severe diarrhea.

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Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.01316-17 ◽

2017 ◽

Vol 56 (3) ◽

Cited By ~ 13

Author(s):

M. J. T. Crobach ◽

N. Duszenko ◽

E. M. Terveer ◽

C. M. Verduin ◽

E. J. Kuijper

Keyword(s):

Nucleic Acid ◽

Clostridium Difficile ◽

Characteristic Curve ◽

Nucleic Acid Amplification Test ◽

Nucleic Acid Amplification ◽

Toxin A ◽

Toxin B ◽

Content Type ◽

Quantitative Results ◽

Testing Algorithm

ABSTRACT Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples ( n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle ( C q ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean C q values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of C q values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal C q cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, C q values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

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Differential expression of TLRs 2, 4, 9, iNOS and TNF-α and arginase activity in peripheral blood monocytes from glucantime unresponsive and responsive patients with anthroponotic cutaneous leishmaniasis caused by Leishmania tropica

Microbial Pathogenesis ◽

10.1016/j.micpath.2018.11.004 ◽

2019 ◽

Vol 126 ◽

pp. 368-378 ◽

Cited By ~ 8

Author(s):

Razieh Tavakoli Oliaee ◽

Iraj Sharifi ◽

Ali Afgar ◽

Abdollah Jafarzadeh ◽

Amir Tavakoli Kareshk ◽

...

Keyword(s):

Cutaneous Leishmaniasis ◽

Differential Expression ◽

Peripheral Blood ◽

Arginase Activity ◽

Leishmania Tropica ◽

Blood Monocytes ◽

Peripheral Blood Monocytes ◽

Tnf Α

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Preferential Induction of TNF-α and IL-1β and Inhibition of IL-10 Secretion by Human Peripheral Blood Monocytes by Synthetic Aza–Alkyl Lysophospholipids

Cellular Immunology ◽

10.1006/cimm.1999.1457 ◽

1999 ◽

Vol 193 (2) ◽

pp. 125-133 ◽

Cited By ~ 4

Author(s):

Xiao-Hu Gan ◽

Benjamin Bonavida

Keyword(s):

Peripheral Blood ◽

Human Peripheral Blood ◽

Blood Monocytes ◽

Peripheral Blood Monocytes ◽

Tnf Α

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Sensitivity of Single-Molecule Array Assays for Detection of Clostridium difficile Toxins in Comparison to Conventional Laboratory Testing Algorithms

Journal of Clinical Microbiology ◽

10.1128/jcm.00452-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 11

Author(s):

Alice Banz ◽

Aude Lantz ◽

Brigitte Riou ◽

Agnès Foussadier ◽

Mark Miller ◽

...

Keyword(s):

Clostridium Difficile ◽

Single Molecule ◽

Laboratory Diagnosis ◽

Case Definition ◽

Cell Cytotoxicity ◽

Toxin A ◽

Toxin B ◽

Content Type ◽

High Performing ◽

Molecular Tests

ABSTRACT Guidelines recommend the use of an algorithm for the laboratory diagnosis of Clostridium difficile infection (CDI). Enzyme immunoassays (EIAs) detecting C. difficile toxins cannot be used as standalone tests due to suboptimal sensitivity, and molecular tests suffer from nonspecificity by detecting colonization. Sensitive immunoassays have recently been developed to improve and simplify CDI diagnosis. Assays detecting CD toxins have been developed using single-molecule array (SIMOA) technology. SIMOA performance was assessed relative to a laboratory case definition of CDI defined by positive glutamate dehydrogenase (GDH) screen and cell cytotoxicity neutralizing assay (CCNA). Samples were tested with SIMOA assays and a commercial toxin EIA to compare performance, with discrepancy resolution using a commercial nucleic acid-based test and a second cell cytotoxicity assay. The SIMOA toxin A and toxin B assays showed limits of detection of 0.6 and 2.9 pg/ml, respectively, and intra-assay coefficients of variation of less than 10%. The optimal clinical thresholds for the toxin A and toxin B assays were determined to be 22.1 and 18.8 pg/ml, respectively, with resultant sensitivities of 84.8 and 95.5%. In contrast, a high-performing EIA toxin test had a sensitivity of 71.2%. Thus, the SIMOA assays detected toxins in 24% more samples with laboratory-defined CDI than the high performing toxin EIA (95% [63/66] versus 71% [47/66]). This study shows that SIMOA C. difficile toxin assays have a higher sensitivity than currently available toxin EIA and have the potential to improve CDI diagnosis.

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