MAPPING OF THE ANTIBODY-BINDING REGIONS ON BOTULINUM NEUROTOXIN H-CHAIN DOMAIN-855-1296 WITH ANTITOXIN ANTIBODIES FROM 3 HOST SPECIES

Botulism due to food poisoning is caused mainly by protein toxins, bot ulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and p oisons known. BoNT effects blockade of neuromuscular transmission by p reventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunizati on with a 43-kDa C-terminal fragment (H-c, residues 860-1296) of BoNT/ A affords excellent protection against BoNT/A poisoning. We raised ant ibodies (Abs) against BoNT/A in horse, and against pentavalent toroid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecuti vely by 5 residues, and encompassed the entire length of the H, of BoN T/A were synthesized and used for mapping the Ab-binding regions recog nized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855-873, 939-957, 1079-1097/1093-1111 overlap, 1191-1209/ 1205-1223 overlap, 1261-1279 and 1275-1296. In addition, peptides 883- 901, 911-929, 995-1013, 1023-1041/1037-1055 overlap, 1121-1139, and 11 49-1167 gave low, but significant and reproducible, binding. With huma n antisera, high amounts of Abs were bound by peptides 869-887, 925-94 3, 981-999, 995-1013, 1051-1069, and 1177-1195. In addition, lower amo unts of Abs were bound by peptides 911-929, 939-957, 967-985, and the overlaps 1121-1139/1135-1153 and 1247-1265/1261-1279/1275-1296. With o utbred mouse antisera, high amounts of Abs were bound by peptides 869- 887, 1051-1069, and 1177-1195, while peptides 939-957, 995-1013, 1093- 1111, and 1275-1296 bound lower amounts of Abs. The results indicate t hat horse antiserum against BoNT/A or human and mouse (outbred) antise ra against the toroid recognized similar regions on BoNT/A, but exhibi ted some boundary frame shifts and differences in immunodominance of t hese regions among the antisera. Selected synthetic epitopes will be u sed as immunogens to stimulate active or passive (by Ab transfer) immu nity against toxin poisoning.