Production and Evaluation of a Recombinant Chimeric Vaccine against Clostridium botulinum Neurotoxin Types C and D

Abstract

Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

“…Thus, the development of recombinant chimeras containing two or more antigens has been spotlighted, either in immunoprophylaxis of clostridial, or other diseases [106,107,108]. The work of Zeng et al [26] evaluated the capacity of two vaccine formulations, rCPA plus rCPB2B1 and a trivalent chimera rCPAB2B1, to generate neutralizing antitoxin in the serum and colostrum of swine and bovine.…”

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry

“…Technically challenging is the fact that, depending on the fragment analysed, BoNT/C, D, and their mosaic variants show a high degree of identity on the protein level, 65 as summarised in Fig. [69][70][71][72] Only BoNT/C and BoNT/DC were commercially available as purified proteins from C. botulinum culture supernatants (see Experimental), but BoNT/D and BoNT/CD were important as well for differential immunisation, screening, and for validation studies. 45,66,67 While this allowed the application of sufficient amounts of antigen to trigger an immune response, the altered 3-dimensional structure often resulted in antibodies preferentially recognising the denatured toxoid over the native toxin.…”

Structure–function discrepancy inClostridium botulinumC3 toxin for its rational prioritization as a subunit vaccine

“…Novel recombinant chimera vaccine consisting of Escherichia coli heat-labile enterotoxin B subunit and the HC of BoNTs C and D has produced for the control of botulism serotypes C and D in cattle (Gil et al, 2013;Krüger et al, 2013;Cunha et al, 2014). Site-directed mutagenesis was conducted in ADP-ribosyltransferase catalytic center for the development of cholera toxin A gene-mediated adjuvant for protein-based vaccines (Wan et al, 2014).…”