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.

Humoral responses in cattle to commercial vaccines containing Clostridium perfringens epsilon toxoid and C. botulinum types C and D toxoids last less than a-year.

The aim of this study was to evaluate the titers of neutralizing antibodies in cattle inoculated with multivalent commercial clostridial vaccines containing C. botulinum type C (BoNTC), C. botulinum type D (BoNTD), and C. perfringens epsilon (ETX) toxoids for a period of one year. Cattle (Bos taurus), aged 4-6 months and not previously immunized, were vaccinated under four different protocols at days 0 and 30 and followed over one year. Individual serum titration was performed by a serum neutralization test in mice or in MDCK cells. The number of animals with detectable neutralizing antibodies ranged from 40.6% to 78.1%, but only 12.5% of animals showed neutralizing antibodies against all tested antigens. Neutralizing antibodies were found only until 60 days for ETX, 120 days for BoNTC, and 180 days for BoNTD. The absence of detectable neutralizing antibodies against the three antigens before 360 days, suggests that cattle remained unprotected for a long period before the recommended booster vaccination.

Evaluation of Adenylate Cyclase Toxoid Antigen in Acellular Pertussis Vaccines by Using a Bordetella pertussis Challenge Model in Mice.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTaA14Q-dmLT or CFA/I/II/IV-2xSTaN12S-dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STaN12S, and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa+ or LT+ ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa+ ETEC and LT+ ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines.

Toxoid Vaccination against Bacterial Infection Using Cell Membrane-Coated Nanoparticles.

As nanoparticles exhibit unique properties attractive for vaccine development, they have been progressively implemented as antigen delivery platforms and immune potentiators. Recently, cell membrane-coated nanoparticles have provided a novel approach for intercepting and neutralizing bacterial toxins by leveraging their natural affinity to cellular membranes. Such toxin-nanoparticle assemblies, termed nanotoxoids, allow rapid loading of different types of toxins and have been investigated for their ability to effectively confer protection against bacterial infection. This topical review will cover the current progress in antibacterial vaccine nanoformulations and highlight the nanotoxoid platform as a novel class of nanoparticulate vaccine. We aim to provide insights into the potential of nanotoxoids as a platform that is facile to implement and can be broadly applied to help address the rising threat of super pathogens.

Active immunization of cattle with a bothropic toxoid does not abrogate envenomation by Bothrops asper venom, but increases the likelihood of survival.

This study assessed the protective effect of active immunization of cattle to prevent the envenomation induced by B. asper venom. Two groups of oxen were immunized with a bothropic toxoid and challenged by an intramuscular injection of either 10 or 50 mg B. asper venom, to induce moderate or severe envenomations, respectively. Non-immunized oxen were used as controls. It was found that immunized oxen developed local edema similar to those observed in non-immunized animals. However, systemic effects were totally prevented in immunized oxen challenged with 10 mg venom, and therefore antivenom treatment was not required. When immunized oxen were challenged with 50 mg venom, coagulopathy was manifested 3-16 h later than in non-immunized oxen, demonstrating a delay in the onset of systemic envenomation. In these animals, active immunization did not eliminate the need for antivenom treatment, but increased the time lapse in which antivenom administration is still effective. All experimentally envenomed oxen completely recovered after a week following venom injection. Our results suggest that immunization of cattle with a bothropic toxoid prevents the development of systemic effects in moderate envenomations by B. asper, but does not abrogate these effects in severe envenomation.

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.

[Mechanisms of formation of post-vaccinal immune response in children immunized with APDT and ADT-M preparations].

AIM: Study the mechanisms of formation of cell and humoral immunity against pertussis, diphtheria and tetanus in children immunized with immunobiological preparations (APDT vaccine and ADT anatoxin). MATERIALS AND METHODS: 30 practically healthy children (6 - 9 years of age) immunized with APDT and ADT-M preparations had TLR2, TLR4 expression determined in mononuclear cells (MNC). Vaccine preparations (APDT, ADT-M, AD-M, AT) and Corynebacterium diphtheriae gravis tox+, C. diphtheriae mitis tox- and Bordetella pertussis 345 were used as ligands. Cytokine production was determined in EIA. Content of anti-diphtheria, anti-tetanus and anti-pertussis antibodies--by PHA reaction and EIA. RESULTS: During stimulation with vaccines and B. pertussis 345 strain MNC were characterized by an increase (p < 0.05) of expression level of TLR2 and TLR4 and did not respond to stimulation with C. diphtheriae gravis tox+ and C. diphtheriae mitis tox- strains. Similar results were obtained during study of cytokine production (TNFalpha, IL-1, IL-6). A direct correlation between levels of antitoxic antibodies against diphtheria and tetanus (R = 0.486), antibacterial antibodies against pertussis and diphtheria was detected (R = 0.529). CONCLUSION: Analysis of cytokine production profile and determination of surface TLR expression can be used during evaluation of functional status of innate immunity cells and intensity of post-vaccinal immunity.

A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile.

The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.

Maternal immunization with vaccines containing recombinant NetB toxin partially protects progeny chickens from necrotic enteritis.

Avian necrotic enteritis is a major economic and welfare issue throughout the global poultry industry and is caused by isolates of Clostridium perfringens that produce NetB toxin. Previously we have shown that birds directly vaccinated with inactivated C. perfringens type A culture supernatant (toxoid) combined with recombinant NetB (rNetB) protein were significantly protected from homologous and heterologous challenge. In the present study the protective effect of maternal immunization was examined. Broiler breeder hens were injected subcutaneously with genetically toxoided rNetB(S254L) alone, C. perfringens type A toxoid and toxoid combined with rNetB(S254L). Vaccination resulted in a strong serum immunoglobulin Y response to NetB in hens immunized with rNetB(S254L) formulations. Anti-NetB antibodies were transferred to the eggs and on into the hatched progeny. Subclinical necrotic enteritis was induced experimentally in the progeny and the occurrence of specific necrotic enteritis lesions evaluated. Birds derived from hens immunized with rNetB(S254L) combined with toxoid and challenged with a homologous strain (EHE-NE18) at either 14 or 21 days post-hatch had significantly lower levels of disease compared to birds from adjuvant only vaccinated hens. In addition, birds from hens immunized with rNetB(S254L) alone were significantly protected when challenged at 14 days post-hatch. These results demonstrate that maternal immunization with a NetB-enhanced toxoid vaccine is a promising method for the control of necrotic enteritis in young broiler chickens.

Vaccination of turkeys with Clostridium septicum bacterin-toxoid: evaluation of protection against clostridial dermatitis.

Clostridial dermatitis is an acute disease causing high mortality in turkeys. Both Clostridium septicum and Clostridium pefringens have been isolated from these cases; however, reports from several diagnostic laboratories indicate an increased isolation rate of C septicum compared with C. perfringens from cases of clostridial dermatitis in recent years. Previous studies suggested C. septicum was more potent than C. perfringens in causing clostridial dermatitis in turkeys. The objective of this study was to develop and evaluate the use of a C. septicum bacterin-toxoid to control clostridial dermatitis in turkeys. A C. septicum bacterin-toxoid was prepared and was initially tested in 6-wk-old commercial turkeys under laboratory conditions for its safety and efficacy. Subsequently, the bacterin-toxoid was evaluated for use in commercial turkey farms with a consistent history of clostridial dermatitis. Birds in the field were vaccinated subcutaneously once at 6 wk of age with C. septicum bacterin-toxoid, and then mortality in both vaccinated and unvaccinated groups was recorded and compared. Blood samples from birds in both groups were examined using ELISA to detect antibody response to the C. septicum toxoid. The C. septicum bacterin-toxoid was found to be safe and to elicit antibodies against the toxoid. In vaccinated commercial turkeys, control of clostridial dermatitis was achieved via antibiotic use and clostridial dermatitis mortality was significantly reduced compared with that of birds in the unvaccinated group. The C. septicum bacterin-toxoid seems to be a valuable tool for the turkey industry to reduce losses due to clostridial dermatitis.

Efficacy of Clostridium botulinum types C and D toxoid vaccination in Danish cows.

In the present study the efficacy Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of Clostridium botulinum types C and D) was evaluated in four Danish dairy cows under field conditions. Other four dairy herds were unvaccinated. Blood serum of all animals was analyzed for specific C. botulinum types A, B, C, D and E antibodies using a developed ELISA. Feces of all animals were analyzed for botulinum neurotoxins (BoNTs) and C. botulinum spores. C. botulinum types C and D antibodies were significantly (p < 0.05) increased in vaccinated animals. Vaccination with botulism vaccine significantly reduced (p < 0.001) BoNTs and C. botulinum spores in cattle feces. Our findings represent that C. botulinum vaccination increases specific blood serum antibodies and reduces free BoNTs and C. botulinum spores in feces.

Vaccination with parenteral toxoid B protects hamsters against lethal challenge with toxin A-negative, toxin B-positive clostridium difficile but does not prevent colonization.

Toxin A has historically been regarded as the primary virulence determinant in Clostridium difficile infection, but naturally occurring toxin A-negative, toxin B-positive (A-/B+) C. difficile strains are known to be virulent. To determine the role of toxin B in these strains, we immunized hamsters with a toxoid prepared from purified toxin B to determine whether they would be protected from lethal challenge with an A-/B+ strain of C. difficile.

Protection of piglets against Edema disease by maternal immunization with Stx2e toxoid.

Edema disease (ED) in piglets is caused by Shiga toxin Stx2e-producing Escherichia coli. We show that a genetically disarmed Stx2e toxoid is a safe antigen that generates antiserum protecting piglets against the Stx2e toxin. Immunization of suckling piglets with the Stx2e toxoid was safe, had no adverse effects on growth of the piglets, and resulted in effective prevention of edema disease clinical symptoms after challenge with the Stx2e toxin. Our data showed that maternal immunity against the Stx2e toxoid can be transmitted from the vaccinated sows to the piglets via the colostrum. Very high levels of Stx2e-specific serum antibodies persisted in these piglets until 1 month postweaning, bridging the critical period in which the weaned piglets are most susceptible to edema infection. Challenge with Stx2e toxin resulted in clinical signs of edema disease and death of all control piglets from nonimmunized sows, whereas none of the piglets from immunized sows developed clinical signs of ED.

[The immune-enzyme testing of titers of serum anti-botulin antibodies type A during immunization of patients with botulin trianiatoxin].

The article discusses the effectiveness of the technique of immune-enzyme detection of anti-botulin antibodies type A in human blood serum. The presence of antibodies is registered in blood serum after second and subsequent immunization with botulin trianatoxin (titer from 1:400 to 1:3200). The establishment of relationship between the registered titers of anti-botulin antibodies and the activity of serum during protection of white mice from toxin revealed the correlation coefficient between these values as 0.58.

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry's production of vaccines against the action of C. perfringens in equine species.

Safety of Diphtheria and Tetanus Toxoids and Acellular Pertussis (DTaP) Vaccine in Adults in Japan.

Tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine is generally used for booster vaccination of infants in Europe and the United States to avoid increased reactogenicity after diphtheria and tetanus toxoids and acellular pertussis (DTaP) vaccination. However, Japan has extended the use of additional DTaP vaccination without reducing the antigen dose for diphtheria and pertussis in adolescents and adults, despite limited reports on its safety in adults. This prospective, observational, questionnaire-based study investigated the occurrence of adverse events (AEs) following DTaP vaccination between June 2018 and June 2019 in participants aged 10 years or older. Of the 250 eligible participants, 235 (94%) responded regarding AEs. Among them, 133 (56.6%) reported AEs, of which 39 reported systemic AEs (16.6%) and 120 reported local AEs (51.1%) attributed to DTaP vaccination. The incidence of local AEs was markedly higher with DTaP vaccination than with non-DTaP vaccination (51.1% vs. 10.5%), and AEs appeared later (P < 0.01) and lasted longer (P < 0.01) with DTaP vaccination. However, more than 75% of these AEs resolved within 7 days. DTaP vaccination was not associated with any serious AEs. These results indicate that the DTaP vaccine can be widely used as a booster in adults as an alternative to the Tdap vaccine.

Safety and efficacy of a maternal vaccine for the passive protection of broiler chicks against necrotic enteritis.

Necrotic enteritis is a potentially fatal multifactorial disease of chickens, which under commercial conditions is often associated with increased levels of mortality and reduced bird performance. The safety and efficacy of a Clostridium perfringens type A alpha-toxoid (Netvax™) formulated as an oil emulsion was investigated, following maternal immunization of broiler breeder hens, housed under commercial conditions, by the intramuscular route. A total of 11,234 hens were vaccinated across two integrated poultry sites. The vaccine was safe with no systemic reactions or adverse effects on bird performance detected. Vaccination resulted in a significant increase in anti-alpha toxin antibody in the hen that was maintained throughout the study, and subsequently transferred to their progeny throughout the laying period via egg yolk. Chicks hatched from eggs produced from vaccinated hens were shown to have reduced mortality specifically related to progeny flocks where gross gut lesions associated with necrotic enteritis were observed in control chicks. Further, whilst C. perfringens was isolated from control chicks with necrotic enteritis lesions, no such isolations were made at these time points from chicks from vaccinated hens. These results indicate that, under commercial conditions, maternal vaccination with Netvax™ can help to control losses related to necrotic enteritis.

Crystalline cholera toxin and toxoid.

The exo-enterotoxin of Vibrio cholerae has been obtained in crystalline form. A solution of the crystalline protein was equal in potency to the parent pure toxin in both choleragenicity and skin reactivity. Crystals of the natural toxoid, choleragenoid, resemble those of the toxin in appearance. A solution of crystalline choleragenoid was equivalent to the parent preparation in the flocculation test.

Virus-like particle-display of the enterotoxigenic Escherichia coli heat-stable toxoid STh-A14T elicits neutralizing antibodies in mice.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.