126An Optimized, Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium difficile Induces Protective Antibody Responses In 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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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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A Replicating Single-Cycle Adenovirus Vaccine Effective against Clostridium difficile

Vaccines ◽

10.3390/vaccines8030470 ◽

2020 ◽

Vol 8 (3) ◽

pp. 470

Author(s):

William E. Matchett ◽

Stephanie Anguiano-Zarate ◽

Goda Baddage Rakitha Malewana ◽

Haley Mudrick ◽

Melissa Weldy ◽

...

Keyword(s):

Clostridium Difficile ◽

Neutralizing Antibodies ◽

Antibody Responses ◽

Complete Protection ◽

Single Cycle ◽

Toxin B ◽

Toxin Binding ◽

Binding Domains ◽

Binding Antibody

Clostridium difficile causes nearly 500,000 infections and nearly 30,000 deaths each year in the U.S., which is estimated to cost $4.8 billion. C. difficile infection (CDI) arises from bacteria colonizing the large intestine and releasing two toxins, toxin A (TcdA) and toxin B (TcdB). Generating humoral immunity against C. difficile’s toxins provides protection against primary infection and recurrence. Thus, a vaccine may offer the best opportunity for sustained, long-term protection. We developed a novel single-cycle adenovirus (SC-Ad) vaccine against C. difficile expressing the receptor-binding domains from TcdA and TcdB. The single immunization of mice generated sustained toxin-binding antibody responses and protected them from lethal toxin challenge for up to 38 weeks. Immunized Syrian hamsters produced significant toxin-neutralizing antibodies that increased over 36 weeks. Single intramuscular immunization provided complete protection against lethal BI/NAP1/027 spore challenge 45 weeks later. These data suggest that this replicating vaccine may prove useful against CDI in humans.

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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 novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models

Vaccine ◽

10.1016/j.vaccine.2012.04.045 ◽

2012 ◽

Vol 30 (28) ◽

pp. 4249-4258 ◽

Cited By ~ 50

Author(s):

Jing-Hui Tian ◽

Steven R. Fuhrmann ◽

Stefanie Kluepfel-Stahl ◽

Robert J. Carman ◽

Larry Ellingsworth ◽

...

Keyword(s):

Fusion Protein ◽

Receptor Binding ◽

Protective Immunity ◽

Lethal Toxin ◽

Toxin A ◽

Toxin B ◽

Binding Domains ◽

Preclinical Efficacy

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Protective antibody responses againstClostridium difficileelicited by a DNA vaccine expressing the enzymatic domain of toxin B

Human Vaccines & Immunotherapeutics ◽

10.4161/hv.22434 ◽

2013 ◽

Vol 9 (1) ◽

pp. 63-73 ◽

Cited By ~ 21

Author(s):

Ke Jin ◽

Shixia Wang ◽

Chunhua Zhang ◽

Yanling Xiao ◽

Shan Lu ◽

...

Keyword(s):

Dna Vaccine ◽

Antibody Responses ◽

Toxin B ◽

Protective Antibody

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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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Effect of purified Clostridium difficile toxins on intestinal smooth muscle. II. Toxin B

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.256.4.g767 ◽

1989 ◽

Vol 256 (4) ◽

pp. G767-G772

Author(s):

R. J. Gilbert ◽

C. Pothoulakis ◽

J. T. LaMont

Keyword(s):

Smooth Muscle ◽

Clostridium Difficile ◽

Membrane Depolarization ◽

Intestinal Smooth Muscle ◽

Toxin A ◽

Toxin B ◽

Direct Exposure ◽

In Vitro Exposure

In the companion paper [Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G759-G766, 1989] we showed that highly purified Clostridium difficile toxin A had a profound effect on intestinal smooth muscle after in vivo but not in vitro exposure. In this study we assessed the effects of in vivo and in vitro exposure to C. difficile toxin B on simultaneous measurements of intracellular membrane potential and contractility in rabbit ileal smooth muscle. Direct exposure of ileal smooth muscle to toxin B (0.1-60 micrograms/ml) in vitro caused membrane depolarization and inhibition of action potential frequency, amplitude, and peak voltage, but no effect on slow wave frequency or amplitude was seen. Toxin exposure also resulted in inhibition of the amplitude of carbachol-induced contractions, with phasic contractions being significantly more sensitive to the effect of toxin B than tonic contractions over the complete dose range. The electromechanical effects of toxin B were not affected by prior administration of tetrodotoxin, atropine, hexamethonium, or phentolamine. In contrast, toxin B administered in vivo into an isolated ileal loop had no effect on spontaneous electromechanical properties of excised smooth muscle strips. Our results indicate that direct exposure in vitro of ileal smooth muscle to C. difficile toxin B causes membrane depolarization in association with inhibition of electromechanical activity. This effect, in combination with the indirect effects of toxin A, may contribute to altered intestinal motility during diarrhea caused by C. difficile.

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