A non-toxic recombinant Clostridium septicum α toxin induces protective immunity in mice and rabbits.

Clostridium septicum alpha toxin (CSA) plays significant roles in ruminant's braxy. Genetically engineered CSA has been shown to function as a potential vaccine candidate in the prevention of the disease caused by Clostridium septicum. In the present study, we synthesized a non-toxic recombinant, rCSAm4/TMD by introducing four amino acid substitutions (C86L/N296A/H301A/W342A) and 11-amino-acid deletion (residues 212 to 222). Compared to recombinant CSA, rCSAm4/TMD showed no cytotoxicity to MDCK cells and was not fatal to mice. Moreover, rCSAm4/TMD could protect immunized mice against 5 × mouse LD100 (100% lethal dose) of crude CSA without obvious pathological change. Most importantly, rabbits immunized with rCSAm4/TMD produced high titers of neutralizing antibodies which protected the rabbits against crude CSA challenge. These data suggest that genetically detoxified rCSAm4/TMD is a potential subunit vaccine candidate against braxy.

Clostridium perfringens epsilon prototoxin mutant rpETXY30A/Y71A/H106P/Y196A as a vaccine candidate against enterotoxemia.

Epsilon toxin (ETX) is secreted by Clostridium perfringens (C. perfringens)as a relatively inactive prototoxin (pETX), which is enzymatically activated to ETX by removing carboxy-terminal and amino-terminal peptides. Genetically engineered ETX mutants have been shown to function as potential vaccine candidates in the prevention of the enterotoxemia caused by C. perfringens. In the present study, two recombinant site-directed mutants of pETX, rpETXY30A/Y71A/H106P/Y196A (rpETXm41) and rpETXY30A/H106P/Y196A/F199E (rpETXm42), were synthesized by mutating four essential amino acid residues (Tyr30, Tyr71, His106, Tyr196 or Phe199). Compared to recombinant pETX (rpETX), both rpETXm41 and rpETXm42 lacked the detectable toxicity in MDCK cells and mice, which suggested that both rpETXm41 and rpETXm42 are sufficiently safe to be vaccine candidates. Despite the fact that rpETXm41 and rpETXm42 were reactogenic with polyclonal antibodies against crude ETX, both single- and double-dose vaccination (Vs and Vd, respectively) of rpETXm41 induced a higher level of IgG titer and protection in mice than that of rpETXm42. Therefore, we selected rpETXm41 for the further study. Sheep received Vs of 150 µg rpETXm41 developed significant levels of toxin-neutralizing antibodies persisting for at least 6 months, which conferred protection against crude ETX challenge without microscopic lesions. These data suggest that genetically detoxified rpETXY30A/Y71A/H106P/Y196A could form the basis of a next-generation enterotoxemia vaccine.

Construction of a Triple-Gene Deletion Mutant of Orf Virus and Evaluation of Its Safety, Immunogenicity and Protective Efficacy.

Contagious ecthyma is a zoonotic disease caused by the orf virus (ORFV). Since there is no specific therapeutic drug available, vaccine immunization is the main tool to prevent and control the disease. Previously, we have reported the construction of a double-gene deletion mutant of ORFV (rGS14ΔCBPΔGIF) and evaluated it as a vaccine candidate. Building on this previous work, the current study reports the construction of a new vaccine candidate, generated by deleting a third gene (gene 121) to generate ORFV rGS14ΔCBPΔGIFΔ121. The in vitro growth characteristics, as well as the in vivo safety, immunogenicity, and protective efficacy, were evaluated. RESULTS: There was a minor difference in viral replication and proliferation between ORFV rGS14ΔCBPΔGIFΔ121 and the other two strains. ORFV rGS14ΔCBPΔGIFΔ121 induced continuous differentiation of PBMC to CD4+T cells, CD8+T cells and CD80+CD86+ cells and caused mainly Th1-like cell-mediated immunity. By comparing the triple-gene deletion mutant with the parental strain and the double-gene deletion mutant, we found that the safety of both the triple-gene deletion mutant and the double-gene deletion mutant could reach 100% in goats, while the safety of parental virus was only 50% after continually observing immunized animals for 14 days. A virulent field strain of ORFV from an ORF scab was used in the challenge experiment by inoculating the virus to the hairless area of the inner thigh of immunized animals. The result showed that the immune protection rate of triple-gene deletion mutant, double-gene mutant, and the parental virus was 100%, 66.7%, and 28.6%, respectively. In conclusion, the safety, immunogenicity, and immune-protectivity of the triple-gene deletion mutant were greatly improved to 100%, making it an excellent vaccine candidate.