Mapping of the Antibody and T Cell Recognition Profiles of the H_N Domain (Residues 449–859) of the Heavy Chain of Botulinum Neurotoxin A in Two High-Responder Mouse Strains

Abstract

Using a set of synthetic overlapping peptides, encompassing the entire N-terminal domain (H_N,) of the heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A), we have mapped on H_N, the regions recognized by Abs (B cells) and by T cells in two inbred mouse strains. After one BoNT/A toxoid injection, BALB/c T cells mounted a weak in vitro response to a region within overlap 687–705/701–719. The remaining peptides stimulated no detectable responses. After 3 injections, BALB/c T cells gave stronger responses to an expanded region within the overlap 687–705/701–719/715–733, peaking at 701–719. BoNT/A-primed BALB/c T cells showed substantial cross-reaction with BoNT/B but did not respond to TeNT. Unlike BALB/c T cells, BoNT/A-primed T cells of SJL cross-reacted well with both BoNT/B and with TeNT. They also recognized a lager epitope profile than the corresponding BALB/c T cells. After one injection with BoNT/A toxoid, SJL T cells responded in vitro to a number of the H_N peptides. Regionswithin peptides 617–635 and 561–579 stimulated strong in vitro responses. Several peptides (463–481, 589–607, 659–677, 729–747, 827–845, and 841–859 revoked weak-to-medium proliferative activities. Four other peptides stimulated very low but reproducible responses (SI between 2.0 and 3.0). After 3 BoNT/A injections, SJL T cells responded in vitro strongly to peptides 463–481, 561–579, 617–635, 743–761, and 841–859. There were medium or weak responses to at least 10 other peptides. The cells also responded well to the l-chain peptide 218–231. Antisera of BALB/c and SJL, obtained after 3 injections with BoNT/A toxoid, protected at very high dilutions recipient mice against LD₁₀₅ of BoNT/A. BALB/c Abs showed medium-to-high binding to peptides 533–551/547–565, 785–803, and 813–831/827–845. Four other peptides showed very low binding. The corresponding SJL Abs had high binding to the overlap 533–551/547–565/561–579, and peptides 743–761, 785–803, and 813–831. Three other peptides bound low amounts of Abs. The results indicate that the responses to each Ab or T cell epitope is under separate genetic control and that, in a given strain, the Ab and T cell recognition regions may coincide but, in addition, H_N contains regions that are recognized only by Abs or only by T cells.

• Botulinum neurotoxin A
• H_N domain
• Antibody recognition regions
• T cell recognition regions
• Epitopes
• Synthetic peptides
• Genetic restriction

Abbreviations
Ab:	=	antibody
BoNT:	=	botulinum neurotoxin
BoNT/A:	=	botulinum neurotoxin serotype A
BSA:	=	bovine serum albumin
CFA:	=	complete Freund's adjuvant
H chain:	=	the heavy chain (residues 449–1296) of BoNT/A
HN:	=	The N-terminal domain (residues 449–859) of the H chain of BoNT/A
HC:	=	C-terminal domain (residues 855–1296) of the H chain of BoNT/A
LNC:	=	lymph node cells
PBS:	=	0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.20
SI:	=	stimulation index