Investigation into immunological responses against a native recombinant CTB whole-cell Vibrio cholerae vaccine in a rabbit model

Abstract

Aim: The aim of this study was to express and purify the recombinant CTB (rCTB) protein from Vibrio cholerae and investigate the biological and immunological characteristics of purified protein in rabbit animal model and in combination with Iranian inactivated V. cholerae whole cells as a domestic recombinant WC-CTB vaccine. Methods and Results: Expressed 6XHis-tagged rCTB was properly purified, and its identity was confirmed by Western blotting using cholera toxin-specific antibody. Concentration of purified protein was assessed to be 700 mg l À1 .GM 1 -ELISA assay showed that purified rCTB pentamer was functionally active and able to bind GM 1 in a dose-dependent manner. Recombinant CTB was inoculated into rabbits through intestinal rout alone and in combination with inactivated whole-cell V. cholerae strains (WC). The anti-CTB IgG titre showed that serum IgG responses were significantly increased in groups immunized with rCTB mixed with inactivated WC in comparison with control group. Furthermore, rCTB without V. cholerae WC also stimulated the IgG responses when inoculated into rabbit intestine. Challenge experiments of immunized rabbits showed an adequate protection against V. cholerae strains. Conclusions: Recombinant CTB alone and in combination with inactivated Iranian strains was protective against live toxigenic V. cholerae strains, made it a potential candidate for an indigenous vaccine. Significance and Impact of the Study: It was proved that rCTB produced in this system can be used as a potent immunogenic protein to stimulate the immunity against V. cholerae strains and can be used for developing a native vaccine composed of our local strains with their own surface structures and antigenic determinants against cholera.

A Single Point Mutation within the Coding Sequence of Cholera Toxin B Subunit Will Increase Its Expression Yield

“…Our previously published data have indicated the pQE- ctx B construct as a highly efficient system to produce rCTB [34] and high affinity binding to GM1 ganglioside [35]. Some advantages of this construct are as follow: i) pQE30 contains the T5 promoter that is recognized by the E. coli host RNA polymerase.…”

The hows and whys of constructing a native recombinant cholera vaccine

“…In addition, rCTB was shown to stimulate the immune response using a rabbit model. 14 Our previous results showed that two different ctxB genotypes were present among V. cholerae isolated from recent outbreaks in Iran. 25 The circulation of inhabitant strains in a region can underline the significance of developing a potent local vaccine composed of our domestic strains with their own cholera toxin antigenic specifications for the control and prevention of cholera outbreaks in this region.…”

“…Recently, we introduced a potent expression system consisting of BL21 (DE3) plus pAE_ctxB construct as an expression vector and have compared it with other expression systems. 14,18 Our previous study demonstrated that expression of rCTB in pAE_ctxB construct is more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. 18 The yield of recombinant CTB produced within this construct in E.coli BL21 (DE3) was about 700 mg l -1…”

“…5,6,8,10,11 These are single dose vaccines, which can elicit a high titer of serum vibriocidal antibodies because they can actively colonize the host intestinal lumen. 6,[12][13][14] The major defect of the engineered live vaccine strains is the probability of acquiring the enterotoxin gene through horizontal gene transfer. This, in turn, can transform the strains carrying toxins into virulent strains which could be troublesome, especially in epidemic areas.…”

A plant-made cholera toxin B subunit enhances mucosal wound healing and protects against ulcerative colitis and colon cancer.

“…We should also point out that the adjuvant potential of CTB has also been shown in large animal models, indicating that the adjuvant potential is scalable to higher species (48)(49)(50).…”