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Antigenic determinants are often composed of discontiguous polypeptide segments, adjacent on protein surfaces, which are difficult to map by conventional techniques. Here we show that although monoclonal antibodies did not recognize recombinant molecules containing only the aminoterminal or only the carboxyterminal part of a protein, after in vitro refolding they are able to recognize a mixture of the two molecules. This suggests that two molecules, each containing part of a discontiguous epitope, are able to reconstitute in vitro the conformation recognized by a monoclonal antibody. Mapping of the regions required for antibody binding can therefore be achieved by in vitro refolding of recombinant molecules. Site-directed mutagenesis is then used to further define the sequences involved in antibody recognition. This new approach was used to map a discontiguous, conformational epitope on the S1 subunit of pertussis toxin. The analysis revealed that pertussis toxin is likely to contain only one immunodominant epitope, which is recognized by all seven protective monoclonal antibodies available to us. In theory, the method described can be used to map conformational epitopes in any protein.

the introduction of two amino acid substitutions within the enzymatically active subunit S1 of pertussis toxin (PT) abolishes its ADP-ribosyltransferase activity and toxicity on CHO cells (Pizza et al., Science 246:497-500, 1989). These genetically inactivated molecules are also devoid of other in vivo adverse reactions typical of PT, such as induction of leukocytosis, potentiation of anaphylaxis, stimulation of insulin secretion, and histamine sensitivity. However, the mutant PT molecules are indistinguishable from wild-type PT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maintain all the physical and chemical properties of PT, including affinity for toxin-neutralizing poly- and monoclonal antibodies. Either alone or stabilized with formaldehyde, PT mutants are able to induce high levels of neutralizing antibodies and to protect mice in a dose-dependent fashion against intracerebral challenge with virulent B. pertussis. These results clearly show that these genetically inactivated PT molecules are nontoxic but still immunogenic and justify their development as a component of a new, safer acellular vaccine against whooping cough.

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.