A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A

Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated and then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to C. difficile toxin A.     

Adenovirus-based vaccination against Clostridium difficile toxin A allows for rapid humoral immunity and complete protection from toxin A lethal challenge in mice

Clostridium difficile associated diarrhea (CDAD) is a critical public health problem worldwide with over 300,000 cases every year in the United States alone. Clearly, a potent vaccine preventing the morbidity and mortality caused by this detrimental pathogen is urgently required. However, vaccine efforts to combat C. difficile infections have been limited both in scope as well as to efficacy, as such there is not a vaccine approved for use against C. difficile to date. In this study, we have used a highly potent Adenovirus (Ad) based platform to create a vaccine against C. difficile. The Ad-based vaccine was able to generate rapid and robust humoral as well as cellular (T-cell) immune responses in mice that correlated with provision of 100% protection from lethal challenge with C. difficile toxin A. Most relevant to the clinical utility of this vaccine formulation was our result that toxin A specific IgGs were readily detected in plasma of Ad immunized mice as early as 3 days post vaccination. In addition, we found that several major immuno-dominant T cell epitopes were identified in toxin A, suggesting that the role of the cellular arm in protection from C. difficile infections may be more significant than previously appreciated. Therefore, our studies confirm that an Adenovirus based-C. difficile vaccine could be a promising candidate for prophylactic vaccination both for use in high risk patients and in high-risk environments.     

Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB₍₀₂₇₎). TcdB₍₀₂₇₎ is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB₍₀₀₃₎). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB₍₀₀₃₎, TcdB₍₀₂₇₎ and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB₍₀₀₃₎/TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB₍₀₀₃₎/TcdA/TcdB₍₀₂₇₎) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI).     

Expression of Clostridium perfringens epsilon-beta fusion toxin gene in E. coli and its immunologic studies in mouse

Clostridium perfringens is an anaerobic spore-forming, pathogenic bacterium that is responsible for severe diseases in humans and livestock. In the present study, an epsilon-beta fusion toxin was expressed as a soluble protein in E. coli and the recombinant cell lysate was used for immunization studies in mouse. Potency of the toxin (as an antigen) induced 6 and 10 IU/ml of epsilon and beta anti-toxin in rabbit, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia for epsilon and beta toxins. Experimental challenge with the recombinant fusion toxoid revealed that it could protect mice against C. perfringens epsilon and beta toxins. Toxicity of the fusion toxin was studied by histopathological findings, which were the same as the native toxins. In conclusion, E. coli is a suitable expression host for immunogenic epsilon-beta fusion toxin of C. perfringens.     

Detection of Clostridium sordellii strains expressing hemorrhagic toxin (TcsH) and implications for diagnostics and regulation of veterinary vaccines

Clostridium sordellii is a Gram positive anaerobic bacterium that causes multiple disease syndromes in both humans and animals. As with many clostridial pathogens, toxins contribute to the virulence of C. sordellii. Two large toxins have been identified: a lethal toxin (TcsL) and a hemorrhagic toxin (TcsH) which are similar in structure and function to Clostridium difficile toxin B (TcdB) and toxin A (TcdA), respectively. While TcdA, TcdB, and TcsL have been extensively studied, relatively little is known about TcsH. This study elucidated the TcsH gene sequence using whole genome sequencing, compared the genotype with toxin expression of 52 C. sordellii strains, and examined the role of TcsH in batch release potency tests required for veterinary vaccines licensed in the United States and other testing utilizing WHO standard antitoxin. Data from this study will assist in future research to clarify the TcsH contribution to the pathogenesis of C. sordellii infections and may aid in the development of improved vaccines.     

Potential protective immunogenicity of recombinant Clostridium perfringens α-β2-β1 fusion toxin in mice, sows and cows

Clostridial toxins are main pathogenic virulence of Clostridium perfringens that have been associated with a wide range of diseases in both humans and domestic animals. Genetically engineered toxoids have been shown to function as potential vaccine candidates in the prevention of Clostridium derived infectious diseases. In this study, we have developed recombinant α-toxin (CPA), β2/β1-fusion toxin (CPB2B1) and α/β2/β1 trivalent fusion-toxin (CPAB2B1) as vaccine candidates that may be used to vaccinate against C. perfringens α, β1 and β2-toxins. Mice immunized with these recombinant toxoids demonstrated a strong protective immunological response when administered a lethal dose of C. perfringens type C culture filtrate with high titers of neutralizing antibodies to the toxins in the sera, as well as the intestinal mucosal s-IgA level. Specific neutralizing antibodies to the toxins were also detected in the sera and colostrum of sows and cows vaccinated with the toxoids. Furthermore, the CPA and CPB2B1 recombinant toxoid cocktail was capable of stimulating relatively higher levels of immune responses compared to that of CPA, CPB2B1 and CPAB2B1 alone. The CPAB2B1 trivalent fusion toxoid also displayed increased immunogenicity relative to CPA and CPB2B1 alone. These results suggest that recombinant toxoids are potential vaccine candidates against Clostridial toxins; the use of mixed cocktails and/or multivalent recombinant toxoids against different types of toxins may be an effective approach in the prevention of diseases caused by toxins produced by C. perfringens.     

Design of antibody-reactive peptides from discontinuous parts of scorpion toxins

The Amm VIII protein was previously isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus. Despite 87% identity with AaH II, the most toxic α-type scorpion toxin, Amm VIII is not toxic to mice. However, antisera against Amm VIII protect mice from AaH II lethal action. Here, we report that the Amm VIII protein elicits antibodies that only recognize discontinuous-type epitopes since we could not observe any antibody binding to overlapping 12-mer peptides covering the whole Amm VIII sequence. By using a new bioinformatic tool, 24 peptides mimicking discontinuous regions of Amm VIII were designed in silico, then prepared by Spot synthesis. Seven of these discontinuous–continuous peptides were recognized by anti-Amm VIII antibodies. Analysis of the 3D location of the segments that compose the antigenically reactive discontinuous–continuous peptides, allowed us to group those antigenic segments into three regions of Amm VIII, putatively corresponding to discontinuous antigenic regions of α-type scorpion toxins. Anti-Amm VIII antibodies were also found to cross-react towards several of the discontinuous–continuous peptides designed from the AaH II structure, pointing to a possible involvement of the corresponding discontinuous epitopes in the capacity displayed by anti-Amm VIII antibodies to neutralize AaH II. Altogether, our results show that it is possible to design antibody-reactive peptides from discontinuous parts of scorpion toxins. The position of the reactive segments in the structural context of scorpion toxins highlights the antigenic properties of the Amm VIII anatoxin and concurs to explain the capacity of anti-Amm VIII antibodies to neutralize the potent AaH II toxin.     

Heterologous protection against alpha toxins of Clostridium perfringens and Staphylococcus aureus induced by binding domain recombinant chimeric protein

Clostridium perfringens and Staphylococcus aureus are the two important bacteria frequently associated with majority of the soft tissue infections. The severity and progression of the diseases caused by these pathogens are attributed primarily to the alpha toxins they produce. Previously, we synthesized a non-toxic chimeric molecule r-αCS encompassing the binding domains of C. perfringens and S. aureus alpha toxins and demonstrated that the r-αCS hyperimmune polysera reacts with both the native wild type toxins. In the present report, we evaluated efficacy of r-αCS in conferring protection against C. perfringens and S. aureus alpha toxin infections in murine model. Immunization of BALB/c with r-αCS was effective in inducing both high titers of serum anti-r-αCS antibodies after three administrations. Sub-typing the antibody pool revealed high proportions of IgG1 indicating a Th2-polarized immune response. The r-αCS stimulated the proliferation of splenocytes from the immunized mice upon re-induction by the antigen, in vitro. The levels of interleukin-10 increased while TNF-α was found to be downregulated in the r-αCS induced splenocytes. Mice immunized with r-αCS were protected against intramuscular challenge with 5 × LD₁₀₀ doses of C. perfringens and S. aureus alpha toxins with >80% survival, which killed control animals within 48–72 h. Passive immunization of mice with anti-r-αCS serum resulted in 50–80% survival. Our results indicate that r-αCS is a remarkable antigen with protective efficacy against alpha toxin mediated C. perfringens and S. aureus soft tissue co-infections.     

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT₅₀) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD₅₀ dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.     

Binary toxin and death after Clostridium difficile infection

We compared 30-day case-fatality rates for patients infected with Clostridium difficile possessing genes for toxins A and B without binary toxin (n = 212) with rates for patients infected with C. difficile possessing genes for A, B, and binary toxin. The latter group comprised patients infected with strains of PCR ribotype 027 (CD027, n = 193) or non-027 (CD non-027, n = 72). Patients with binary toxin had higher case-fatality rates than patients without binary toxin, in univariate analysis (relative risk [RR] 1.8, 95% confidence interval [CI] 1.2-2.7) and multivariate analysis after adjustment for age, sex, and geographic region (RR 1.6, 95% CI 1.0-2.4). Similar case-fatality rates (27.8%, 28.0%) were observed for patients infected with CD027 or CD non-027. Binary toxin either is a marker for more virulent C. difficile strains or contributes directly to strain virulence. Efforts to control C. difficile infection should target all virulent strains irrespective of PCR ribotype.     

Active and passive immunization against Clostridium difficile diarrhea and colitis

Clostridium difficile, a gram-positive bacterium, is the major cause of hospital-acquired infectious diarrhea and colitis in industrialized nations. C. difficile colonization results from antibiotic administration and subsequent loss of protection provided by intestinal flora. C. difficile induced-colitis is caused by the release of two exotoxins, toxin A and B. Host factors including advanced age, pre-existing severe illness and weakened immune defenses predispose individuals to symptomatic infection. The generation of antibody responses to toxin A through natural exposure is associated with protection from disease. In addition, an inability to acquire immunity to toxin A puts individuals at risk for recurrent and/or severe disease. Immunological approaches for the management of this disease are being developed which could reduce the reliance on antibiotics for treatment and allow for re-establishment of the natural barrier provided by an intact commensal flora. An active vaccine and various immunotherapeutic strategies under evaluation may prove to be effective against severe or relapsing C. difficile infection.     

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process.     

Proteomic profiling of precipitated Clostridioides difficile toxin A and B antibodies

Clostridioides difficile infection is the leading cause of nosocomial diarrhoea globally. Immune responses to toxins produced by C. difficile are important in disease progression and outcome. Here, we analysed the anti-toxin A and anti-toxin B serum antibody proteomes following natural infection or vaccination with a C. difficile toxoid A/toxoid B vaccine using a modified miniaturised proteomic approach based on de novo mass spectrometric sequencing. Analysis of immunoglobulin variable region (IgV) subfamily expression in immunoprecipitated toxin A and toxin B antibodies from four and seven participants of a vaccine trial, respectively, revealed a polyclonal proteome with restricted IGHV, IGKV and IGLV subfamily usage. No dominant IGHV subfamily was observed in the toxin A response, however the dominant anti-toxin B heavy (H)-chain was encoded by IGHV3-23. Light (L)-chain usage was convergent for both anti-toxin A and anti-toxin B proteomes with IGKV3-11, 3-15, 3-20 and 4-1 shared among all subjects in both cohorts. Peptide mapping of common IgV families showed extensive public and private amino acid substitutions. The cohort responses to toxin A and toxin B showed limited similarity in shared IGHV subfamilies. L-chain subfamily usage was more similar in the anti-toxin A and anti-toxin B responses, however the mutational signatures for each subfamily were toxin-dependent. Samples taken both post vaccination (n = 5) or at baseline, indicating previous exposure (n = 2), showed similar anti-toxin B IgV subfamily usage and mutational profiles. In summary, this study provides the first sequence-based proteomic analysis of the antibody response to the major disease-mediating toxins of C. difficile, toxin A and toxin B, and demonstrates that despite the potential for extreme diversity, the immunoglobulin repertoire can raise convergent responses to specific pathogens whether through natural infection or following vaccination.     

Vaccine-induced intestinal immunity to ricin toxin in the absence of secretory IgA

The RNA N-glycosidase ribosome inactivating proteins (RIPs) constitute a ubiquitous family of plant- and bacterium-derived toxins that includes the category B select agents ricin, abrin and shiga toxin. While these toxins are potent inducers of intestinal epithelial cell death and inflammation, very little is known about the mechanisms underlying mucosal immunity to these toxins. In the present study, we report that secretory IgA (SIgA) antibodies are not required for intestinal immunity to ricin, as evidenced by the fact that mice devoid of SIgA, due to a mutation in the polymeric immunoglobulin receptor, were impervious to the effects of intragastric toxin challenge following ricin toxoid immunization. Furthermore, parenteral administration of ricin-specific monoclonal IgGs, directed against either ricin's enzymatic subunit (RTA) or ricin's binding subunit (RTB), to wild type mice was as effective as monoclonal IgAs with comparable specificities in imparting intestinal immunity to ricin. These data are consistent with reports from others demonstrating that immunization of mice by routes known not to induce mucosal antibody responses (e.g., intramuscular and intradermal) is sufficient to elicit protection against both systemic and mucosal ricin challenges.     

Deciphering the domain specificity of C. difficile toxin neutralizing antibodies

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. The pathological effects of CDI are primarily attributed to toxins A (TcdA) and B (TcdB). Adequate toxin-specific antibody responses are associated with asymptomatic carriage, whereas insufficient humoral responses are associated with recurrent CDI. While the data supporting the importance of anti-toxin antibodies are substantial, clarity about the toxin domain specificity of these antibodies is more limited. To investigate this matter, combinations of human mAbs targeting multiple domains of TcdB were assessed using toxin neutralization assays. These data revealed that a combination of mAbs specific to all major toxin domains had improved neutralizing potency when compared to equivalent concentrations of a single mAb or a combination of mAbs against one or two domains. The function and toxin domain binding specificity of serum antibodies elicited by immunization of hamsters with a toxoid vaccine candidate was also assessed. Immunization with a toxoid vaccine candidate provoked toxin neutralizing antibodies specific to multiple domains of both TcdA and TcdB. When assessed in a toxin neutralization assay, polyclonal sera displayed greater activity against elevated concentrations of toxins than equivalent concentrations of individual mAbs. These data suggest a potential benefit of any antibody based therapeutic or prophylactic treatment that targets multiple toxin domains.     

Clostridium difficile: development of a novel candidate vaccine

Clostridium difficile has become the most frequent hospital-acquired infection in North America and the EU. C. difficile infection (CDI) is present worldwide and disease awareness is increasing. In the US, EU, and Canada, in addition to hospital diagnosed disease, CDI has also been reported with increasing frequency in the community. Hypervirulent strains have increased the morbidity and mortality associated with CDI. Current treatment options are suboptimal. Of all patients treated for CDI, 20% relapse and 65% of those experiencing a second relapse become chronic cases. An association between increased serum levels of IgG antibody against toxin A and asymptomatic carriage of C. difficile provides a rationale for vaccine development. Sanofi Pasteur's C. difficile candidate vaccine is being developed for the prevention of primary disease. The target population is adults at risk of CDI, those with planned hospitalization, long-term care/nursing home residents, and adults with co-morbidities requiring frequent/prolonged antibiotic use.     

A two-stage algorithm for Clostridium difficile including PCR: can we replace the toxin EIA?

A two step, three-test algorithm for Clostridium difficile infection (CDI) was reviewed. Stool samples were tested by enzyme immunoassays for C.?difficile common antigen glutamate dehydrogenase (G) and toxin A/B (T). Samples with discordant results were tested by polymerase chain reaction detecting the toxin B gene (P). The algorithm quickly identified patients with detectable toxin A/B, whereas a large group of patients excreting toxigenic C.?difficile but with toxin A/B production below detection level (G(+)T(-)P(+)) was identified separately. The average white blood cell count in patients with a G(+)T(+) result was higher than in those with a G(+)T(-)P(+) result.     

Clostridium difficile infection in patients with haematological malignant disease. Risk factors, faecal toxins and pathogenic strains

Two hundred and forty-eight patients from shared oncology and general medical wards were prospectively studied over a 6-month period for carriage of Clostridium difficile during an outbreak of clinical disease with an epidemic strain of the organism. Risk factors for infection were assessed. Acute leukaemia and/or its treatment were identified as significantly increasing the risk of infection. The relationship between the type of C. difficile isolated (as defined by a typing system based on the incorporation of [35S]methionine into bacterial proteins followed by gel electrophoresis), the presence of faecal toxins A and B and clinical symptoms were analysed. Carriage of the epidemic strain, type X, had a significant association with symptoms amongst oncology patients, with two thirds of these patients having detectable faecal toxin A and one third detectable faecal toxin B. During an outbreak of C. difficile-associated disease, typing the organism and assaying for both faecal toxins in symptomatic patients may be of benefit in determining which patients require specific, urgent treatment.     

Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit

Peptides KDYGASTGSSL (Epil). SLLRRRRNGEDVSV (Epi3) and DDEIYGNDGHP (Epi6), predicted to constitute immunogenic epitopes of the hemolysin-cytotoxin ApxIA of Actinobacillus pleuropneumoniae were inserted into a surface-exposed loop of the B subunit of the E. coli heat-labile enterotoxin (EtxB). The resulting chimeric proteins were recognized by monospecific antibodies against purified native ApxI and by convalescent sera of pigs that were positive for A. pleuropneumoniae serotype 1. Mice anti-sera against chimeric proteins EtxB::ApxIAEpi3 and EtxB::ApxIAEpi6 reacted with purified ApxI. These results indicate that Epi3 and Epi6 regions constitute linear epitopes of the structural ApxIA protein toxin. Epitope Epi6 which is located in the structure of the glycine rich repeats in ApxI elicits the formation of hemolysin neutralizing antibodies when introduced into mice in the form of a chimeric EtxB fusion protein. We suggest that fusion of peptide sequences to EtxB is a useful tool for the analysis of epitopes of complex proteins such as RTX toxins.     

Expression of a poliovirus type 1 neutralization epitope on a diphtheria toxin fusion protein

The diphtheria toxin (DT) secreted by Corynebacterium diphtheriae is used after formolization as an efficient vaccine against diphtheria. In an attempt to evaluate its capacity to present heterologous peptide sequence in a recognized form, we created in-phase insertion in the gene encoding the non-toxic mutant protein CRM228 of DT. The sequence chosen for insertion was the synthetic DNA fragment encoding a poliovirus neutralization epitope. Tripartite fusion proteins comprising the mutant DT, the poliovirus peptide and beta-galactosidase were obtained in E. coli and purified by affinity chromatography. These fusion proteins reacted both with antibodies directed against the DT and a poliovirus specific monoclonal antibody. Moreover, these hybrid toxins induced protective antibodies against the lethal effect of DT and neutralizing antibodies against poliovirus. We conclude that the modification of highly immunogenic DT may provide a means for the presentation of foreign peptide sequences to the immune system.