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.

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

“…Further studies have focused on determining the toxicity mechanism of CPB and have generated important recommendations for vaccinology specialists that have facilitated the development of recombinant vaccines [61,62,63,64]. Of the various strategies that are used to develop recombinant vaccines containing CPB, four approaches, in particular, are worth highlighting: (1) the insertion of point mutations for the generation of toxoids; (2) the expression of the whole toxin sequence; (3) the expression of its C -terminal domain (CPB-C (143–311) ); and (4) the expression of chimeric antigens containing other toxins (e.g., CPA, CPB2, or ETX) or the B subunit of the heat-labile enterotoxin of E. coli (LTB) (Table 3) [18,26,65,66,67,68,69,70,71]. …”

“…Since treatment is not a feasible option, this increases the need for a prevention method, mainly via vaccination, that can avoid the loss of animals. The main approaches for the development of a recombinant vaccine against ETX are the use of rETX-carrying mutations to eliminate its toxicity, the use of Lactobacillus casei to carry these mutated antigens to its surface, the expression of the protoxin (rPETX), and the use of chimeras that contain multiple antigens (Table 4) [20,22,69,71,80,95,96,97,98]. …”

“…The prime immunization was performed with rPETX associated with Freund’s complete adjuvant (FCA) and boosted with Freund's incomplete adjuvant (FIA). Moreira et al [69] synthesized a CDS of ETX without the sequence for the 45 amino acids at the N -terminal portion using optimized codons for expression in E. coli and obtained a soluble rPETX that was non-toxic for MDCK cells. Rabbits vaccinated with 200 µg of rCPA, rCPB, and rPETX co-administered with Al(OH) 3 as adjuvant showed titers of 25 IU/mL of antitoxin ETX.…”

“…Numerous studies have described the use of recombinant toxins in illness immunoprophylaxis caused by C. perfringens in food animals (Table 5) [22,26,68,69,94,104]. Jiang et al [105] evaluated the treatment of calves using a trivalent vaccine that contained 300 µg of each antigen (rCPA (247–370) , rCPB and rETX) emulsified in oil adjuvant ISA 15A VG.…”

Optimization of recombinant vaccine antigen dose in mouse model by ELISA and immunophenotyping

“…In order to improve vaccine efficacy, rational preclinical development of recombinant antigens and improved protocols for predicting the immunogenicity of antigens is an important endeavour. The efficacy of majority of the vaccine candidates prepared from purified recombinant antigens and peptides depends on the antigens alone, the success of subunit vaccines also depends on the adjuvants added to vaccine formulations to boost the immune response to the antigen [5,6]. Undoubtedly, investigation of the effect of dose of antigens on murine models before vaccine development is an important goal.…”