An Optimized, Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium difficile Induces Protective Antibody ResponsesIn Vivo

ABSTRACTClostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clearC. difficileinfection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putativeN-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed byin vivoelectroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in anin vitrocytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged withC. difficilespores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.

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126An Optimized, Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium difficile Induces Protective Antibody Responses In Vivo

Open Forum Infectious Diseases ◽

10.1093/ofid/ofu051.34 ◽

2014 ◽

Vol 1 (suppl_1) ◽

pp. S12-S12

Author(s):

Michele Kutzler

Keyword(s):

Clostridium Difficile ◽

Dna Vaccine ◽

Receptor Binding ◽

Antibody Responses ◽

Toxin A ◽

Toxin B ◽

Protective Antibody ◽

Synthetic Dna ◽

Binding Domains

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Vaccination againstClostridium difficileby Use of an AttenuatedSalmonella entericaSerovar Typhimurium Vector (YS1646) Protects Mice from Lethal Challenge

Infection and Immunity ◽

10.1128/iai.00089-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 1

Author(s):

Kaitlin Winter ◽

Li Xing ◽

Audrey Kassardjian ◽

Brian J. Ward

Keyword(s):

Clostridium Difficile ◽

Receptor Binding ◽

Lethal Challenge ◽

Content Type ◽

Binding Domains ◽

Mucosal Antibody ◽

Serovar Typhimurium ◽

Antibody Titers ◽

Further Development ◽

Detectable Serum

ABSTRACTClostridium difficiledisease 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 optimalC. difficilevaccine would generate both systemic and mucosal responses. We have repurposed an attenuatedSalmonella entericaserovar 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 C57BL/6J mice. When given in a multimodality schedule over 1 week (intramuscularly and orally [p.o.] on day 0 and p.o. on days 2 and 4), several candidates provided 100% protection against lethal challenge. Substantial protection (82%) was achieved with combined p.o. TcdA and TcdB vaccination alone (days 0, 2, and 4). These data demonstrate the potential of the YS1646-based vaccines forC. difficileand strongly support their further development.

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Immunogenicity and Protection from Receptor-Binding Domains of Toxins as Potential Vaccine Candidates for Clostridium difficile

Vaccines ◽

10.3390/vaccines7040180 ◽

2019 ◽

Vol 7 (4) ◽

pp. 180

Author(s):

Deyan Luo ◽

Xuechao Liu ◽

Li Xing ◽

Yakun Sun ◽

Jie Huang ◽

...

Keyword(s):

Clostridium Difficile ◽

Recombinant Proteins ◽

Receptor Binding ◽

Vaccine Development ◽

Toxin A ◽

Toxin B ◽

Vaccine Candidates ◽

Humoral Immune ◽

Binding Domains ◽

New Type

The receptor-binding domains (RBDs) located in toxin A and toxin B of Clostridium difficile are known to be nontoxic and immunogenic. We need to develop a new type vaccine based on RBDs. In this study, we expressed and purified recombinant proteins (named RBD-TcdA and RBD-TcdB) as vaccine candidates containing the RBDs of toxin A and toxin B, respectively, from the C. difficile reference strain VPI10463. The immunogenicity and protection of the vaccine candidates RBD-TcdA, RBD-TcdB, and RBD-TcdA/B was evaluated by ELISA and survival assays. The data indicated that mice immunized with all vaccine candidates displayed potent levels of RBD-specific serum IgG. Following intramuscular immunization of mice with RBD-TcdA and/or RBD-TcdB, these vaccine candidates triggered immune responses that protected mice compared to mice immunized with aluminum hydroxide alone. Taken together, the results of this study reveal that recombinant proteins containing RBDs of C. difficile toxins can be used for vaccine development. Additionally, we found that an RBD-TcdA/B vaccine can elicit a stronger humoral immune response and provide better immunoprotection than the univalent vaccines. This RBD vaccine candidate conferred significant protection against disease symptoms and death caused by toxins from a wild-type C. difficile strain.

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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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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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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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Vaccination With Parenteral Toxoid B Protects Hamsters Against Lethal Challenge With Toxin A–Negative, Toxin B–Positive Clostridium difficile but Does Not Prevent Colonization

The Journal of Infectious Diseases ◽

10.1093/infdis/jir688 ◽

2011 ◽

Vol 205 (1) ◽

pp. 128-133 ◽

Cited By ~ 22

Author(s):

Farida Siddiqui ◽

Jennifer R. O’Connor ◽

Kristin Nagaro ◽

Adam Cheknis ◽

Susan P. Sambol ◽

...

Keyword(s):

Clostridium Difficile ◽

Toxin A ◽

Lethal Challenge ◽

Toxin B

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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

Infection and Immunity ◽

10.1128/iai.00011-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 194-204 ◽

Cited By ~ 6

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.

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