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 se rotype 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 w ith one injection of toroid, recognized two major regions within resid ues 897-915 and 939-957. After multiple inoculations with toroid, T ce lls of Balb/c expanded their recognition ability and responded very we ll to challenge with peptide 1261-1279 and moderately to stimulation w ith peptide 1249-1167. Unlike Balb/c T cells, those of toroid-primed S JL (H-2(S)) mice exhibited a more complex profile and responded to cha llenge with a large number of overlapping peptides. After one toroid i njection, however, three peptides, 897-925, 939-957/953-971 overlap an d 1052-1069, were the most potent T cells stimulators. After three tor oid injections, peptides 897-915 and 1051-1069 remained immunodominant while the third region was shifted upstream to 925-943/939-957 overla p. The immunodominant epitope within peptide 897-915 was recognized ex clusively by T cells, since no Abs were detected against this region. The Ab binding profiles of the two mouse strains were quite similar, s howing only small quantitative differences. Both, Balb/c and SJL anti- toroid Abs displayed strong binding mainly to peptide 2177-1195, follo wed 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 995-101 3 and 1052-1069, the anti-toroid Abs of SJL mice exhibited strong bind ing 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.