Loratadine is an anti-inflammatory agent against C. difficile toxin B.

BACKGROUND: Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical non-microbiological therapeutic approach against CDI. METHODS: We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI10463 and A-B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B-dependent pathways in PBMCs. RESULTS: Histamine 1 receptor-related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein one alpha (MIP-1α) expression in PBMCs. We identified Loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B-induced MIP-1α secretion in fresh human colonic tissues. Oral Loratadine (10 mg/kg/day) maintained survival, inhibited intestinal Ccl3 mRNA expression, and prevented vancomycin-associated recurrence in the VPI10463-infected mice and ribotype 017-infected hamsters. Splenocytes from Loratadine-treated mice conferred anti-inflammatory effects to the VPI10463-infected T/B cell-deficient Rag-/- mice. Oral Loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. CONCLUSIONS: Loratadine may be repurposed to optimize existing therapies against CDI.

Loratadine is an FDA-approved antihistamine that inhibits toxin B-mediated pro-inflammatory macrophage inflammatory protein one alpha secretion from immune cells. The anti-inflammatory effect of Loratadine ameliorates intestinal inflammation in C. difficile-infected animals. Loratadine may be repurposed to optimize existing therapies.

Genistein Inhibits Clostridioides difficile Infection via Estrogen Receptors and Lysine-Deficient Protein Kinase 1.

BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

A fine-tuned yeast surface-display/secretion platform enables the rapid discovery of neutralizing antibodies against Clostridioides difficile toxins.

BACKGROUND: Neutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19. RESULTS: In this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable 'switch' through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB. CONCLUSION: The antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Delivery of a therapeutic antibody to the lower gastrointestinal tract for the treatment of Clostridium difficile infection (CDI).

The colonic delivery system of toxin neutralizing antibody is a promising method for treating Clostridium difficile infection (CDI) and has some advantages over the parental administration of a neutralizing antibody. However, colonic delivery of biologics presents several challenges, including instability of biologics during encapsulation into the delivery system and harsh conditions in the upper GI tract. In this work, we described a multi-particulate delivery system encapsulating a tetra-valent antibody ABAB-IgG1 with the potential to treat CDI. This work first approved that the cecum injection of ABAB-IgG1 into the lower GI tract of mice could relieve the symptoms, enhance the clinical score, and improve the survival rate of mice during CDI. Then, the antibody was spray layered onto mannitol beads and then enteric coated with pH-sensitive polymers to achieve colon-targeting release. The in vitro release of antibody from the multi-particulate system and the pH-sensitive release of antibody was monitored. The in vivo efficacy of this system was further examined and confirmed in mice and hamsters. In summary, the findings of this study should provide practical information and potential treatment options for CDI through colonic delivery of antibody therapeutics to the lower GI tract using a multi-particulate delivery system.

Correction: Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

[This corrects the article DOI: 10.1371/journal.pbio.3000311.].

Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.

Therapeutic Mechanism of Macrophage Inflammatory Protein 1 α Neutralizing Antibody (CCL3) in Clostridium difficile Infection in Mice.

BACKGROUND: Clostridium difficile infection (CDI) causes diarrhea and colitis. We aimed to find a common pathogenic pathway in CDI among humans and mice by comparing toxin-mediated effects in human and mouse colonic tissues. METHOD: Using multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A- and B-treated human and mouse colonic explants. RESULTS: Toxin A and toxin B exposure to fresh human and mouse colonic explants caused different patterns of cytokine secretion. Toxin A induced macrophage inflammatory protein (MIP) 1α secretion in both human and mouse explants. Toxin A reduced the expression of chloride anion exchanger SLC26A3 expression in mouse colonic explants and human colonic epithelial cells. Patients with CDI had increased colonic MIP-1 α expression and reduced colonic SLC26A3 (solute carrier family 26, member 3) compared with controls. Anti-MIP-1 α neutralizing antibody prevented death, ameliorated colonic injury, reduced colonic interleukin 1ß (IL-1ß) messenger RNA expression, and restored colonic SLC26a3 expression in C. difficile-infected mice. The anti-MIP-1 α neutralizing antibody prevented CDI recurrence. SLC26a3 inhibition augmented colonic IL-1 ß messenger RNA expression and abolished the protective effect of anti-MIP-1 α neutralizing antibody in mice with CDI. CONCLUSION: MIP-1 α is a common toxin A-dependent chemokine in human and mouse colon. MIP-1 α mediates detrimental effects by reducing SLC26a3 and enhancing IL-1 ß expression in the colon.

TPL2 Is a Key Regulator of Intestinal Inflammation in Clostridium difficile Infection.

Tumor progression locus 2 (TPL2), a serine/threonine protein kinase, is a major inflammatory mediator in immune cells. The predominant inflammatory actions of TPL2 depend on the activation of mitogen-activated protein kinases (MAPK) and the upregulated production of the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1ß (IL-1ß) in macrophages and dendritic cells in response to lipopolysaccharide (LPS). Significant increases in TNF-α, IL-6, IL-ß, and IL-8 levels in patients with Clostridium difficile infection (CDI) have been reported. Both TNF-α and IL-6 have been postulated to play key roles in the systemic inflammatory response in CDI, and IL-8 is essential for the development of local intestinal inflammatory responses in CDI. The objective of this study was to elucidate the role of TPL2 in the pathogenesis of CDI. We found that TPL2 was significantly activated in human and mouse intestinal tissues upon C. difficile toxin exposure or CDI. We further demonstrated that TPL2 knockout (TPL2-KO) mice were significantly more resistant to CDI than wild-type mice, with significantly reduced production of TNF-α, IL-6, IL-1ß, KC (a mouse homologue of IL-8), and myeloperoxidase (MPO) in the ceca and colons of TPL2-KO mice. Finally, we found that TPL2 inhibition by a specific inhibitor or TPL2 gene ablation significantly reduced TcdB-induced production of TNF-α, IL-6, IL-ß, and KC by inhibiting the activation of p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK). Taken together, our data suggest that TPL2 represents a potential therapeutic target for CDI treatment.

A Multiparticulate Delivery System for Potential Colonic Targeting Using Bovine Serum Albumin as a Model Protein : Theme: Formulation and Manufacturing of Solid Dosage Forms Guest Editors: Tony Zhou and Tonglei Li.

PURPOSE: There are many important diseases whose treatment could be improved by delivering a therapeutic protein to the colon, for example, Clostridium difficile infection, ulcerative colitis and Crohn's Disease. The goal of this project was to investigate the feasibility of colonic delivery of proteins using multiparticulate beads. METHODS: In this work, bovine serum albumin (BSA) was adopted as a model protein. BSA was spray layered onto beads, followed by coating of an enteric polymer EUDRAGIT® FS 30 D to develop a colonic delivery system. The secondary and tertiary structure change and aggregation of BSA during spray layering process was examined. The BSA layered beads were then challenged in an accelerated stability study using International Council for Harmonization (ICH) conditions. The in vitro release of BSA from enteric coated beads was examined using United States Pharmacopeia (USP) dissolution apparatus 1. RESULTS: No significant changes in the secondary and tertiary structure or aggregation profile of BSA were observed after the spray layering process. Degradation of BSA to different extents was detected after storing at 25°C and 40°C for 38 days. Enteric coated BSA beads were intact in acidic media while released BSA in pH 7.4 phosphate buffer. CONCLUSION: We showed the feasibility of delivering proteins to colon in vitro using multiparticulate system.

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.

The Brucella melitensis M5-90 phosphoglucomutase (PGM) mutant is attenuated and confers protection against wild-type challenge in BALB/c mice.

Brucellae are Gram-negative intracellular bacterial pathogens that infect humans and animals, bringing great economic burdens to developing countries. Live attenuated Brucella vaccines (strain M5-90 or others) are the most efficient means for prevention and control of animal brucellosis. However, these vaccines have several drawbacks, including residual virulence in animals, and difficulties in differentiating natural infection from vaccine immunization, which limit their application. A vaccine that can differentiate infection from immunization will have extensive applications. A Brucella melitensis (B. melitensis) strain M5-90 pgm mutant (M5-90Δpgm) was constructed to overcome these drawbacks. M5-90Δpgm showed significantly reduced survival in embryonic trophoblast cells and in mice, and induced high protective immunity in BALB/c mice. Moreover, M5-90Δpgm elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In addition, M5-90Δpgm induced the secretion of IFN-γ in immunized sheep. Serum samples from sheep inoculated with M5-90Δpgm were negative by the Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Furthermore, the PGM antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90Δpgm is an ideal live attenuated vaccine candidate against B. melitensis 16 M and deserves further evaluation for vaccine development.

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.

Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides.

Clostridium difficile toxin A and B (TcdA and TcdB) are the major virulence factors of the bacterium, both of which consist of two enzymatic domains: an effector glucosyltransferase domain (GTD) and a cysteine protease domain (CPD) responsible for autocleavage and release of GTD. Although the CPDs from both toxins share a similar structure and mechanism of hexakisphosphate (InsP6)-induced activation, TcdA is substantially less sensitive to the autocleavage as compared with TcdB. In this study, we provided evidence of inter-domain regulation of CPD activity of TcdA and its autoprocessing. The C-terminus combined repetitive oligo peptides (CROPs) of TcdA reduced the accessibility of TcdB CPD to its substrate in a chimeric toxin TxB-Ar, consequently blocking autoprocessing. Moreover, interference of antibodies with the CROPs of full-length TcdA efficiently enhanced its GTD release. In conclusion, by utilizing chimeric toxins and specific antibodies, we identified that the CROPs of TcdA plays a crucial role in controlling the InsP6-mediated activation of CPD and autocleavage of GTD. Our data provides insights on the molecular mode of action of the C. difficile toxins.

A novel multivalent, single-domain antibody targeting TcdA and TcdB prevents fulminant Clostridium difficile infection in mice.

The incidence of Clostridium difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. In this study, we generated a novel, potently neutralizing, tetravalent, and bispecific antibody composed of 2 heavy-chain-only VH (VHH) binding domains against both TcdA and TcdB (designated "ABA") that reverses fulminant CDI in mice infected with an epidemic 027 strain after a single injection of the antibody. We demonstrated that ABA bound to both toxins simultaneously and displayed a significantly enhanced neutralizing activity both in vitro and in vivo. Additionally, ABA was able to broadly neutralize toxins from clinical C. difficile isolates that express both TcdA and TcdB but failed to neutralize the toxin from TcdA(-)TcdB(+) C. difficile strains. This study thus provides a rationale for the development of multivalent VHHs that target both toxins and are broadly neutralizing for treating severe CDI.

Systemic dissemination of Clostridium difficile toxins A and B is associated with severe, fatal disease in animal models.

BACKGROUND: Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years. METHODS: Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI. RESULTS: We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI. CONCLUSION: Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.

A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection.

The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.

MBX-500, a hybrid antibiotic with in vitro and in vivo efficacy against toxigenic Clostridium difficile.

Clostridium difficile infection (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, the in vitro and in vivo efficacies 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.

Retracted: Apoptosis of CT26 colorectal cancer cells induced by Clostridium difficile toxin A stimulates potent anti-tumor immunity.

Clostridium difficile toxin A (TcdA) is one of the main pathogenic factors released by C. difficile. Due to its potent cytotoxic and proinflammatory activities, we investigated the anti-tumor activity of TcdA. CT26 colorectal cancer cells were challenged with recombinant TcdA, and it was found that TcdA could induce apoptosis of CT26 cells. Calreticulin (CRT) exposure to the cell surface during TcdA-induced apoptosis suggested that this apoptosis may correlate with immunogenicity. Moreover, TcdA-treated apoptotic CT26 cells were highly immunogenic since they could stimulate DC activation, T-cell activation, and anti-tumor activity. Furthermore, the anti-tumor immune response generated was specific and long-term. In summary, these studies demonstrate that C. difficile toxin A can induce apoptotic death of CT26 colorectal cancer cells and stimulate potent anti-tumor immunity.