A recombinant mutant abrin A chain expressed in Escherichia coli can be used as an effective vaccine candidate.

We used site-directed mutagenesis to mutate two key amino acid residues, Glu164 and Arg167, of abrin A chain (ABRA), creating a mutant ABRA(E164AR167L). The mutant ABRA(mABRA) encoded by mABRA(E164AR167L) was expressed in the cytoplasm of Escherichia coli, and used to develop an effective vaccine to protect mice against native abrin intoxication. The cytotoxicity of mABRA was dramatically reduced as compared to that of recombinant ABRA(rABRA) and native abrin, but the antigenicity and immunogenicity remained the same. Balb/c mice were vaccinated with purified mABRA, and survival was evaluated after challenge with native abrin. Mice that were given three vaccinations developed a protective immune response that was 100% protective against an intraperitoneal (i.p.) administration of 10×LD50 of native abrin. Furthermore, the sera from immunized mice provided complete passive protection for naive mice. This study describes the generation of a substantial amount of mABRA from E. coli and the potential application of mABRA as an effective vaccine candidate for humans, to protect against a high-dose of native abrin.

Truncated abrin A chain expressed in Escherichia coli: a promising vaccine candidate.

Abrin toxin (AT) is a highly potent toxin, and is classified as one of the most important biological warfare and bioterrorism agents. There is currently no approved vaccine for AT. Therefore, the development of an effective vaccine is important in the prevention of intoxication by abrin. In this study, five vectors containing different gene of truncated abrin toxin A chain (tATA) fragments were constructed, and two of them (tATA1(1-126), tATA4(1-188)) were successfully expressed as a soluble form in E.coli strain. Both of the two tATA retained most of their immunogenicity with either low or no toxic effects as determined by both in vitro and in vivo assays. They were used to immunize BALB/c mice three times at an interval of three weeks apart. As a result, the tATA1 can elicite 80% protective efficacy against i.p. challenge of 5×LD50 of abrin, and the tATA4 provides a better protection, which can elicite 100% protective efficacy against intraperitoneal challenge of 40×LD50 of abrin. The superior fragment (tATA4(1-188)) should be considered as a promising vaccine candidate for further investigations.

Cloning and expression of three abrin A-chains and their mutants derived by site-specific mutagenesis in Escherichia coli.

DNAs encoding of three abrin A-chains were obtained from the cDNA library of Abrus precatorius by polymerase chain reaction and ligated into the expression vector, pGEX-2T. The mature A-chains of abrins a, b and d have been expressed in the cytoplasm of Escherichia coli, and the yield of the soluble recombinant proteins was 7 mg/l induced culture. Three recombinant abrin A-chains were purified to be homogeneity and their N-glycosidase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The recombinant abrin-a A-chain had the highest N-glycosidase activity among three recombinant abrin A-chains while the recombinant abrin-b A-chain, the least. Three mutants, glutamic-acid-to-alanine replacement (E164A), arginine to leucine (R167L) or double mutation (E164A and R167L) were constructed and expressed. The protein-biosynthesis-inhibitory activity of mutant (E164A), mutant (R167L) and the double mutant was found to be 25-fold, 625-fold and 1250-fold lower than that of wild type, respectively. The results indicated that Arg167 was essential for abrin toxin A-chain catalysis.