Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants

Abstract

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals.

Figure 1. SDS-PAGE 12% and Western blot using anti-6xHis antibody for the detection of rEpsilon.

(A) Solubilization of rEpsilon (38 kDa) expressed in E. coli BL21 (DE3) Star. 1- E. coli BL21 (DE3) Star not transformed; 2- induced E. coli BL21 (DE3) Star transformed with pAE-rEpsilon; 3- supernatant of lysis buffer of induced E. coli BL21 (DE3) Star containing pAE-rEpsilon; 4–BSA (67 kDa). (B) Elution fractions of rEpsilon after Ni^+ 2-affinity purification. 1- supernatant of lysis buffer of induced E. coli BL21 (DE3) Star containing pAE-rEpsilon after purification; 2-3- purified rEpsilon. (C) Western blot using anti-6xHis against purified rEpsilon. 1- PageRuler Prestained Protein Ladder (Thermo Scientific); 2- purified rEpsilon. Of note, (A,C) parts of this figure were cropped from a single image on the dashed lines to be better presented in the article’s context, although the gels have been run under the same conditions and the Western blot performed with the same set of materials. These two complete figures can be found, respectively, as Supplementary Figs S1 and S2.

Figure 2. Antitoxin level generated by model (rabbit) and ruminant (cattle, sheep, and goat) species.

All groups matched or surpassed the minimum level required (dashed line), which are 4, 10, and 2 IU/mL against alpha, beta, and epsilon toxins, respectively. (A) Level of neutralizing antibodies against alpha toxin generated by each of the species. When recombinant and commercial vaccines are compared among the ruminants, cattle and goat showed significant higher levels, but not for sheep. (B) Level of neutralizing antibodies against beta toxin generated by each of the species. Only cattle showed significant higher levels when comparing recombinant and commercial vaccines in farm animals. (C) Level of neutralizing antibodies against epsilon toxin generated by each of the species. By comparing recombinant and commercial vaccines, the former one generated higher antitoxin levels for every ruminant species. *P < 0.05; **P < 0.01.