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We have investigated whether the nonpathogenic gram-positive bacteria Staphylococcus xylosus and S. carnosus can display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE'mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins on S. xylosus and S. carnosus cell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface of S. xylosus cells whereas it is efficiently exposed on S. carnosus. A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally with S. carnosus displaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, only S. carnosus can display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT(168-220)) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT(168-220), and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT(168-220), which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT(168-220) antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies. Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wild-type toxin to AChR. Therefore, transformation of a non-enzymatic toxin into a toxoid can be achieved, like in the case of enzymatic toxins, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by Malpha2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and Malpha2-3 is partial only.