Design of a chitosan-based nano vaccine against epsilon toxin of Clostridium perfringens type D and evaluation of its immunogenicity in BALB/c mice

Abstract

Background and purpose: Clostridium perfringens is an anaerobic, spore-forming, and pathogenic bacterium that causes intestinal diseases in humans and animals. In these cases, therapeutic intervention is challenging; because the disease progresses much rapidly. This bacterium can produce 5 main toxins (alpha, beta, epsilon, iota, and a type of enterotoxin) among which the epsilon toxin (ETX) is used for bioterrorism. This toxin can be prevented by immunization with specific immunogenic vaccines. In the present research, we aimed at developing a recombinant chitosan-based nano-vaccine against ETX of C. perfringens and evaluate its effects on the antibody titration against epsilon toxin in BALB/c mice as the vaccine model.

Experimental approach: The etx gene from C. perfringens type D was cloned and expressed in E. coli. After analysis by SDS-PAGE and western blotting, the expressed products were purified, and the obtained proteins were used for immunization in mice as a chitosan nanoparticle containing recombinant, purified ETX, and protein.

Findings/results: The results of ELISA showed that IgA antibody serum level increased sufficiently using recombinant protein with nanoparticle as an oral and injectable formulation. IgG antibody titers increased significantly after administrating the recombinant proteins with nanoparticles through both oral delivery and intravenous injection.

Conclusion and implication: In conclusion, the recombinant ETX is suggested as a good candidate for vaccine production against diseases caused by ETX of C. perfringens type D.

Keywords: Chitosan; Clostridium perfringens; Epsilon toxin; Immunization; Nano-vaccine.

Fig. 1

(A-C) the genomic DNA extraction, PCR products by primer M13, and the digestion analysis on agarose gel 1%. (A) Lane M: DNA size marker; lane 1: the positive control; and lane 2: etx gene digested by EcoRI and HindIII restriction enzymes. (B) Lane M: DNA size marker and lane 1: etx gene extracted from pBSK. (C) Lane M: DNA size marker and lane 1: digested pET28a by EcoRI and HindIII restriction enzymes. ETX, Epsilon toxin.

Fig. 2

Expression, solubility, and purification of epsilon toxin stained with Coomassie blue on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 10%.

Fig. 3

Western blotting of ETX with specific anti-His tag. Lane M: protein size marker; lane 1: purified recombinant protein ETX; lane 2: non-induced transformed BL21(DE3) as the negative control, and lane 3: pET28a with insert as the positive control. ETX, Epsilon toxin.

Fig. 4

Determination of size and zeta potential of nanoparticles (A) before loading of protein, (B) after loading of protein, and (C) zeta potential after loading of protein.

Fig. 5

IgG serum antibody response of BALB/c mice immunized with recombinant epsilon toxin protein after two doses in (A) oral, (B) oral + injection, (C) injection group, and (D) comparison of oral and oral + injection groups IgG antibody titration. The highest stimulation of antibodies was observed in the oral + injection group. The data represent mean + SD.

Fig. 6

Serum IgA response of BALB/c mice immunized with recombinant epsilon toxin protein after two doses in (A) oral, (B) oral+injection, (C) injection group, and (D) comparison of oral, oral + injection, and injection groups. The highest stimulation of antibodies was observed in the oral group. The data represent mean + SD.

Fig. 7

IgA antibody titration in fecal samples of (A) oral; (B) oral + injection; (C) injection groups, and (D) comparison of oral, oral + injection, and injection fecal groups IgA antibody titration. More stimulation of antibodies was shown in the oral group. The data represent mean + SD.

Fig. 8

Effect of epsilon toxin toxicity on MDCK cells. The data represent mean +SD Phosphate-buffered saline has been used as the negative control group. *P < 0.05 Indicates significant differences compared to the control group.

Fig. 9

Protective effect of sera from immunized mice against MDCK cells. The data represent mean +SD Phosphate-buffered saline has been used as the negative control group. *P < 0.05 Indicates significant differences compared to the control group.