Clostridium perfringens Epsilon Toxin Mutant I51C as a Recombinant Vaccine Candidate Against Enterotoxemia.

BACKGROUND: Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which can lead to enterotoxemia, an extremely lethal disease that has significant consequences for the farming of domestic ruminants, specifically sheep and goats. The bacterin-toxoids/toxoids enterotoxemia vaccines need time-consuming detoxification steps. Genetically derived toxoids (GTs) can be the alternative vaccines against ETX-associated enterotoxemia. This study was aimed to design, synthesize, and evaluate of five epsilon toxin mutants of C. perfringens by site-directed mutagenesis (SDM). METHODS: In this study, five ETX mutants (H106P, I51C, V56C, A114C, and F118C), as ETX-GTs, were designed and synthesized by SDM, which were then cloned in pET-26b (+) and expressed in Escherichia coli /BL21 (DE3). The expression of recombinant ETX-GTs was evaluated by SDS-PAGE, blotting, and ELISA and their toxicity was evaluated by the residual toxicity test based on BP Pharmacopoeia, 2021. RESULTS: The findings showed that the ETX-GTs could be considered alternative vaccine candidates against ETX-associated enterotoxemia. CONCLUSIONS: These data suggest that I51C mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

Clostridium perfringens epsilon prototoxin mutant rpETXY30A/Y71A/H106P/Y196A as a vaccine candidate against enterotoxemia.

Epsilon toxin (ETX) is secreted by Clostridium perfringens (C. perfringens)as a relatively inactive prototoxin (pETX), which is enzymatically activated to ETX by removing carboxy-terminal and amino-terminal peptides. Genetically engineered ETX mutants have been shown to function as potential vaccine candidates in the prevention of the enterotoxemia caused by C. perfringens. In the present study, two recombinant site-directed mutants of pETX, rpETXY30A/Y71A/H106P/Y196A (rpETXm41) and rpETXY30A/H106P/Y196A/F199E (rpETXm42), were synthesized by mutating four essential amino acid residues (Tyr30, Tyr71, His106, Tyr196 or Phe199). Compared to recombinant pETX (rpETX), both rpETXm41 and rpETXm42 lacked the detectable toxicity in MDCK cells and mice, which suggested that both rpETXm41 and rpETXm42 are sufficiently safe to be vaccine candidates. Despite the fact that rpETXm41 and rpETXm42 were reactogenic with polyclonal antibodies against crude ETX, both single- and double-dose vaccination (Vs and Vd, respectively) of rpETXm41 induced a higher level of IgG titer and protection in mice than that of rpETXm42. Therefore, we selected rpETXm41 for the further study. Sheep received Vs of 150 µg rpETXm41 developed significant levels of toxin-neutralizing antibodies persisting for at least 6 months, which conferred protection against crude ETX challenge without microscopic lesions. These data suggest that genetically detoxified rpETXY30A/Y71A/H106P/Y196A could form the basis of a next-generation enterotoxemia vaccine.

A non-toxic recombinant bivalent chimeric protein rETXm3CSAm4/TMD as a potential vaccine candidate against enterotoxemia and braxy.

Clostridium perfringens epsilon toxin (ETX) and Clostridium septicum alpha toxin (CSA) are lethal and necrotizing toxins, which play key roles in enterotoxemia and braxy of ruminants, respectively. In the present study, we synthesized a bivalent chimeric protein rETXm3CSAm4/TMD comprising ETXm3 (Y30A/H106P/Y196A) and CSAm4/TMD (C86L/N296A/H301A/W342A and a deletion of residues 212 to 222). Compared with recombinant ETX and recombinant CSA, rETXm3CSAm4/TMD showed no cytotoxicity in Madin-Darby Canine Kidney cells and was not fatal to mice. Moreover, rETXm3CSAm4/TMD could protect immunized mice against 10 × mouse LD100 of crude ETX or 3 × mouse LD100 of crude CSA without obvious histopathologic difference. Most importantly, both rabbits and sheep immunized with rETXm3CSAm4/TMD produced high titers of neutralizing antibody which protected the animals against the challenge with crude ETX or crude CSA. These data suggest that genetically detoxified rETXm3CSAm4/TMD is a potential subunit vaccine candidate against enterotoxemia and braxy.

A non-toxic recombinant protein rSUMO-CPBm4 as a potential vaccine candidate against Clostridium perfringens type C beta enterotoxemia.

Beta toxin (CPB) is a lethal toxin and plays a key role in enterotoxemia of ruminants caused by Clostridium perfringens type C strain. The existing vaccines based on crude CPB need time-consuming detoxification and difficult quality control steps. In this study, we synthesized the rCPBm4 of C. perfringens type C strain and small ubiquitin-like modifier (SUMO)-tag CPBm4 (rSUMO-CPBm4) by introducing four amino acid substitutions: R212E, Y266A, L268G, and W275A. Compared with rCPBm4, rSUMO-CPBm4 was expressed with higher solubility in Escherichia coli BL21 (DE3). Neither rCPBm4 nor rSUMO-CPBm4 was lethal to mice. Although rCPBm4 and rSUMO-CPBm4 were reactogenic with polyclonal antibodies against crude CPB, rabbits vaccinated with rSUMO-CPBm4 developed significant levels of toxin-neutralizing antibody (TNA) titers that conferred protection against crude toxin challenge. These data suggest that genetically detoxified rSUMO-CPBm4 is a promising subunit vaccine candidate for C. perfringens type C beta enterotoxemia.

A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay.

Enterotoxaemia, a disease that affects domestic ruminants, is caused by the epsilon toxin of Clostridium perfringens type D and B. Control and prophylaxis are based on systemic vaccination of small ruminant herds with epsilon toxoid. Purified epsilon toxin is an essential material for vaccine evaluation. It is also necessary for diagnosis of enterotoxaemia disease in the field by in vitro tests including ELISA. The aim of this study was to set up a method for preparation of functional purified epsilon toxin of C. perfringens type D to be used in serum neutralization test. In this study, epsilon toxin was prepared from C. perfringens type D culture precipitated with ammonium sulfate, dialyzed against phosphate buffered saline (PBS) buffer and then, purified using chromatography system. Then, the purified epsilon toxin was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toxin function was confirmed by cell culture and minimum lethal dose (MLD) assays. Also rabbits were immunized by vaccine in two turns with a 28-day interval. Then, blood samples were collected, and serum neutralization (SN) test was carried out. Results showed that the purified toxin was suitable for SN assay. Our purification method was simple, fast and cost-effective for preparation of epsilon toxin.

Cattle and goats' humoral response to vaccination with Clostridium perfringens type D purified epsilon toxoids.

Herd vaccination is an important preventive measure against enterotoxemia in ruminants. Vaccination in goats should be performed every four months, and recent studies have shown that immunity in cattle lasts for less than one year. One of the mechanisms for increasing the duration of the immune response is to use purified toxoids as immunogens. The aim of the present study was to evaluate the humoral response in cattle and goats after vaccination with purified and semi-purified Clostridium perfringens type D epsilon toxoid. The following three different vaccines were used: vaccine 1 (V1), a semi-purified toxoid adsorbed to aluminum hydroxide; vaccine 2 (V2), a purified toxoid adsorbed to aluminum hydroxide; and vaccine (V3), a purified toxoid adsorbed on chitosan microparticles. Groups of cattle (n = 6-7) and goats (n = 6-7) were vaccinated on days 0 and 30, and serum samples for antitoxin titration were collected every 30 days for one-year post-vaccination. Goats were revaccinated on day 360, and their serum was evaluated on days 367 and 374. The antibody peaks ranged between 6.90 and 11.47 IU/mL in cattle and from 1.11 to 4.40 IU/mL in goats. In cattle administered with the V1 and V2 vaccines, we observed that the antibody titers were maintained above 0.2 IU/mL until the end of the experiment. In goats, V2 elicited long-lasting antibodies, and all animals maintained the protective titers for 210 days after the first dose. In conclusion, the purified toxoid vaccine with aluminum hydroxide adjuvant was able to induce strong and long-lasting humoral responses in both species and could be an alternative for improving the immunization schedule against enterotoxemia in goats and cattle.

Etx-Y71A as a non-toxic mutant of Clostridium perfringens epsilon toxin induces protective immunity in mice and sheep.

Epsilon toxin (Etx) is an extremely potent toxin produced by Clostridium perfringens toxinotypes B and D, which cause fatal enterotoxemia in many livestock species, mainly sheep and goats. Our previous study demonstrated that the aromatic amino acid (AA) residue at position 71 in domain III of Etx is needed for its cytotoxic activity toward MDCK cells. Here, we first determined that Etx mutants with non-aromatic AA substitutions at Tyr71 lost lethality in mice, indicating that the aromatic AA residue at position 71 is a toxicity determinant of Etx in vivo. After intravenous injection with a high dose of the trypsin-activated Etx-Y71A mutant, mice did not show any histopathological lesions, and confocal microscopy observations further showed that Etx-Y71A lost the ability to cross the blood-brain barrier of the mice. These results suggested that the Etx-Y71A mutant is sufficiently safe in vivo to be a vaccine candidate. Furthermore, the immune efficacy of Etx-Y71A was evaluated in model and host animals. Mice inoculated with this mutant produced high levels of neutralizing antibodies and were completely protected from a 100 LD50 of trypsin-activated Etx challenge. Sheep immunized with Etx-Y71A produced high levels of neutralizing antibodies that provided protection in mice against an activated Etx challenge, and lambs could receive passive immunity through immunization of pregnant ewes. Additionally, homology modeling and circular dichroism analysis showed that Etx-Y71A has structural similarity to Etx, which provides a structural basis for Etx-Y71A retaining the immunogenicity of Etx. Taken together, these results suggest that Etx-Y71A is a potential vaccine candidate against Etx-inducing enterotoxemia.

The pore structure of Clostridium perfringens epsilon toxin.

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.

Evaluation of plant-produced Clostridium perfringens type D epsilon toxoid in a vaccine against enterotoxaemia in sheep.

Enterotoxaemia (pulpy kidney) is a common bacterial disease of sheep caused by Clostridium perfringens type D epsilon toxin. It has mortality rates of up to 30% in non-vaccinated animals. Current vaccines from whole cell cultures are expensive to manufacture and can induce local inflammatory responses in sheep. They usually have reduced immunogenicity because of the difficulty of standardising the inactivation step in vaccine manufacturing. In the current study, we evaluated the safety and potency of a recombinant plant-made epsilon toxoid protein (r-Etox) as an affordable and safer alternative vaccine for developing countries. Results of injection site reactions, rectal temperature and toxin neutralisation test in single and prime- boost inoculations of mice, guinea pigs and sheep suggest that the product is not toxic to animals and could protect sheep against enterotoxaemia.

Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX(Y196E)-C confers strong protection in mice.

Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETX(Y196E)-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:10(5) in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 µg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 µg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 µg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 µg) of rETX challenge in mice. These data suggest that recombinant protein rETX(Y196E)-C is a potential vaccine candidate for future applied researches.

Induction of potential protective immunity against enterotoxemia in calves by single or multiple recombinant Clostridium perfringens toxoids.

Cattle enterotoxemia caused by Clostridium perfringens toxins is a noncontagious, sporadic, and fatal disease characterized by sudden death. Strategies for controlling and preventing cattle enterotoxemia are based on systematic vaccination of herds with toxoids. Because the process of producing conventional clostridial vaccines is dangerous, expensive, and time-consuming, the prospect of recombinant toxoid vaccines against diseases caused by C. perfringens toxins is promising. In this study, nontoxic recombinant toxoids derived from α-, ß- and ε-toxins of C. perfringens, namely, rCPA247-370 , rCPB and rEtxHP, respectively, were expressed in Escherichia coli. High levels of specific IgG antibodies and neutralizing antibodies against the toxins were detected in sera from calves vaccinated with either a single recombinant toxoid or a mixed cocktail of all three recombinant toxoids, indicating the potential of these recombinant toxoids to provide calves with protective immunity against enterotoxemia caused by C. perfringens.

Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

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

Epsilon toxin (Etx) is a ß-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.

Modified toxin-binding inhibition (ToBI) test for epsilon antitoxin determination in serum of immunized rabbits.

The aim of the present study was to evaluate and standardize the ToBI test in vitro as a substitute for the serum neutralization test in mice for quality control of clostridial vaccines. The ToBI test in vitro was used to evaluate 40 serum samples of known antibody content, obtained from rabbits immunized against clostridiosis with experimental vaccine. The correlation between epsilon antitoxin titers in rabbit sera, determined by the ToBI test and serum neutralization in mice, ranged from 0.222% to 0.452% in polyvalent vaccines and from 0.154% to 0.387% in monovalent vaccines. Interplate coefficients of variation were not significant, reaching 0.350% in polyvalent vaccines and 0.400% in monovalent vaccines, indicating high homogeneity. In conclusion, the ToBI test in vitro is suitable for assessing the potency of clostridial vaccines and may be used as an alternative method able to replace current in vivo tests.

Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.

Cattle enterotoxaemia and Clostridium perfringens: description, diagnosis and prophylaxis.

Cattle enterotoxaemia is one of numerous pathologies caused by Clostridium perfringens. These anaerobic Gram-positive bacteria are naturally present in the intestinal flora of mammals, but their uncontrolled multiplication under certain conditions results in the overproduction of toxins in the intestinal tract. Major clinical signs are induced by the systemic spread of these toxins in the blood and tissues. Enterotoxaemia may be acute or peracute, and sudden death is often reported in rapidly growing, apparently healthy cattle. Enterotoxaemia can be prevented only with better understanding of its risk factors and pathogenesis. This paper provides an up-to-date overview of knowledge concerning the aetiology of the syndrome, its epidemiological context, pathogenesis, clinical signs and lesions, the diagnostic procedures and prophylactic tools, with specific attention to field aspects that are directly relevant to practitioners and clinical researchers.

Development of a recombinant epsilon toxoid vaccine against enterotoxemia and its use as a combination vaccine with live attenuated sheep pox virus against enterotoxemia and sheep pox.

Sheep pox and enterotoxemia are important diseases of sheep, and these diseases cause severe economic losses to sheep farmers. The present study was undertaken to evaluate the potential of formaldehyde-inactivated recombinant epsilon toxin as a vaccine candidate. The potency of the recombinant epsilon toxoid with aluminum hydroxide as an adjuvant in sheep was determined. Vaccinated sheep were protected against enterotoxemia, with potency values of >5 IU being protective. Further, the use of this construct in a combination vaccine against sheep pox resulted in the sheep being protected against both sheep pox and enterotoxemia.

Antibody response to the epsilon toxin of Clostridium perfringens following vaccination of Lama glama crias.

BACKGROUND: Enterotoxaemia produced by Clostridium perfringens A, C and D is an important cause of mortality in young llamas. There is no data on antibody responses following vaccination with epsilon toxin. METHODOLOGY: Twenty-six L. glama crias were divided into four groups which were vaccinated with a commercial vaccine (Mancha Gangrena Enterotoxemia, Instituto Rosembusch Sociedad Anónima, Argentina) on days 0, 21 and 42 or left as unvaccinated controls. An indirect ELISA was compared with the mouse neutralization test (MNT) for measuring titers to C. perfringens type D epsilon toxin and used to determine titers in sera taken before vaccination and 16, 28, 49, 59, and 93 days later. RESULTS: The ELISA gave comparable results to the MNT and showed animals vaccinated once failed to develop raised titers. A week following a second vaccination, mean antibody titers rose significantly (P < 0.05) and 7/12 animals developed high titers which were present in only one animal at the end of the study (day 93). A third vaccination resulted in a decrease in mean antibody titers a week later. CONCLUSIONS: Llamas develop antibodies to Clostridium perfringens type D epsilon toxin after two vaccinations at a 21-day interval. Further studies are indicated to determine if these inoculations protect against enterotoxemia and the most appropriate vaccination schedule.

Enterotoxemia em bovino / Bovine enterotoxaemia

This report describes a case of bovine enterotoxaemia in Morro da Garça, Minas Gerais, Brazil. Clostridium perfringens type D was isolated in pure culture and was characterized by biochemical reactions and PCR. By the mouse neutralization test, the presence of epsilon toxin from intestinal content was detected.

Immunoprophylactic strategies against enterotoxemia caused by Clostridium perfringens type D in goats

Foi avaliada a resposta sorológica de vacina experimental contra a enterotoxemia em quatro grupos de caprinos. O Grupo 1 recebeu colostro de vacas não vacinadas e nenhuma dose de vacina. Os Grupos 2, 3 e 4 receberam colostro de vacas vacinadas, e uma dose de vacina aos 80 dias de idade nos Grupos 3 e 4. O Grupo 4 recebeu a segunda dose de vacina aos 120 dias de idade. Os níveis de anticorpos séricos foram avaliados pelo ELISA nas vacas antes e depois do parto e nos caprinos aos 3, 80, 120 e 160 dias de idade. Não houve diferença significativa nos níveis de anticorpos séricos das vacas vacinadas e não vacinadas, assim como entre os quatro grupos de caprinos avaliados aos três dias de vida. Os Grupos 3 e 4 apresentaram títulos médios de anticorpos de 0,6 UI/mL e 1,1 UI/mL, respectivamente, aos 40 dias após a primovacinação. A resposta vacinal do grupo 4, 40 dias após o reforço, foi de 1,8 UI/mL, superior ao Grupo 3 que foi de 0,2 UI/mL. Portanto, no esquema proposto, o uso de colostro não induziu a imunização passiva dos cabritos. No entanto, a primovacinação e reforço 40 dias após desencadearam níveis de anticorpos considerados satisfatórios.