Localization of the regions on the C-terminal domain of the heavy chain of botulinum toxin A recognized by T lymphocytes and by antibodies after immunization of mice with pentavalent toxoid

We have mapped the regions recognized by T and/or B cells (Abs) on the C-terminal domain (H-c) of the heavy chain of botulinum neurotoxin serotype A (BoNTIA) after immunization of two inbred mouse strains with pentavalent toroid (BoNTs A, B, C, D and E). Using a set of synthetic overlapping peptides, encompassing the entire H-c domain (residues 855-1296), we demonstrated that T cells of Balb/c (H-2(d)) mice, primed with one injection of toroid, recognized two major regions within residues 897-915 and 939-957. After multiple inoculations with toroid, T cells of Balb/c expanded their recognition ability and responded very well to challenge with peptide 1261-1279 and moderately to stimulation with peptide 1249-1167. Unlike Balb/c T cells, those of toroid-primed SJL (H-2(S)) mice exhibited a more complex profile and responded to challenge with a large number of overlapping peptides. After one toroid injection, however, three peptides, 897-925, 939-957/953-971 overlap and 1052-1069, were the most potent T cells stimulators. After three toroid injections, peptides 897-915 and 1051-1069 remained immunodominant while the third region was shifted upstream to 925-943/939-957 overlap. The immunodominant epitope within peptide 897-915 was recognized exclusively by T cells, since no Abs were detected against this region. The Ab binding profiles of the two mouse strains were quite similar, showing only small quantitative differences. Both, Balb/c and SJL anti-toroid Abs displayed strong binding mainly to peptide 2177-1195, followed by peptides 869-887/883-902 overlap and 2275-1296. In addition, a significant amount of Balb/c anti-toroid Abs was bound to peptide 1235-2153. Unlike Balb/c Abs, that interacted weakly with peptides 995-1013 and 1052-1069, the anti-toroid Abs of SJL mice exhibited strong binding toward both peptides. The results showed that, in a given strain, the regions recognized by anti-toroid Abs and T cells may coincide or may be uniquely B or T cell determinants.