Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge

• Published in: Protein expression and purification Vol. 167; p. 105550
• Main Authors: Peng, Xiaobing, Peng, Guorui, Li, Xuni, Feng, Lifang, Dong, Lingying, Jiang, Yuwen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2020
• Subjects: Alpha toxin; Animals; Antibodies, Bacterial - blood; Bacterial Toxins - biosynthesis; Bacterial Toxins - genetics; Bacterial Toxins - immunology; Bacterial Toxins - isolation & purification; Bacterial Vaccines; Bicistron design system; Calcium-Binding Proteins - biosynthesis; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - immunology; Calcium-Binding Proteins - isolation & purification; Cholera Toxin - genetics; Cloning, Molecular; Clostridium perfringens - genetics; Clostridium perfringens - metabolism; Clostridum perfringens; ELISA; Escherichia coli - genetics; Mice; Rabbits; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant vaccine; Type C Phospholipases - biosynthesis; Type C Phospholipases - genetics; Type C Phospholipases - immunology; Type C Phospholipases - isolation & purification; Vaccination - methods; Clostridum perfringens; Alpha toxin; Bicistron design system; Recombinant vaccine; ELISA
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2019.105550

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247–370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin. •A BCD system for simultaneously expressing rCPA-C(247−370) and CTB-rCPA-C(247−370) is constructed.•The BCD system greatly improves solubility and expression levels of the target proteins.•The antigenic response of expressed products is significantly more protective than the current vaccine in China.•A more stable, high-yielding method with higher biosafety levels to product clostridial antigens is proposed.