Analysis of Epitopes in The Cholera Family of Enterotoxins

Abstract

It is now over 30 years since the cholera enterotoxin was discovered in India and more than 20 years since it was first purified to homogeneity. Early expectations that, as with diphtheria and tetanus, a toxoid vaccine would solve the cholera problem have not been fulfilled. We are examining the reasons why cholera toxin (CT) vaccines have failed to provide effective immunity.

Studies with polyclonal (pAbs) and monoclonal (mAbs) antibodies reveal significant differences among the CTs and CT-related enterotoxins (LTs). All field trials of toxoid vaccines have used CT-1 antigen against the prevailing epitype of CT, usually CT-2. Single amino acid substitutions in the immunodominant B-subunit protein have profound effects on immunological reactivity. We have employed pAbs and mAbs, genetically engineered chimeric B-subunit proteins, checkerboard immunoblotting (CBIB), synthetic peptides and their antisera, and sequential overlapping synthetic hexapeptides representing the B-subunit chain to identify epitopes in the CT family. Reactivity of some, but not all, synthetic hexapeptides with epitope activity with polyclonal antisera can be reduced by pretreatment of the serum with the native toxin protein indicating that certain continuous epitopes are exposed on the surface of the native protein. Interestingly, the holotoxin was more effective in this regard than the native B-subunit pentamer indicating a conformational difference between the two. Human convalescent sera gave diffuse patterns of reactivity with discontinuous (conformational) epitopes. A major tetrapeptide epitope has been identified in a conserved region of the protein. Substitution of amino acids in this region can be permissive or can reduce or eliminate activity. Studies like these may lead to the development of rational synthetic peptide vaccines, but a vaccine composed only of CT antigen can only protect against severe life-threatening diarrhea, not all the diarrhea caused by cholera vibrios.