Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species

Abstract

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (Hc, residues 860-1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the Hc of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized 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 1149-1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869-887, 925-943, 981-999, 995-1013, 1051-1069, and 1177-1195. In addition, lower amounts 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 outbred 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 that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.

Enhancing the Protective Immune Response against Botulism

“…This is consistent with the observation that BoNTs can be cleared by combinations of monoclonal antibodies that do not neutralize toxin activity (59). Furthermore, work by Atassi and his coworkers identified the peptide composing the GPB as a minor yet reproducible immunological epitope, though dominant epitopes varied by species (58).…”

“…In addition, neutralizing antibodies that target the light chain of BoNT/A through another mechanism, potentially by preventing translocation of the LC but not endocytosis of the holotoxin, have been found (54)(55)(56). Numerous epitopes on both the N-and C-terminal regions of HCR/A have been described, and neutralizing activity is absent from antibodies that target the N terminus, distal to the GBP (17,30,57,58).…”

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

“…Atassi and Dolimbek used polyclonal antisera to compare the responses of humans and other animal species to peptides from Botulinum neurotoxin (19,20). These authors observed that antisera from different animals often recognized similar regions on the antigen, but found there were boundary frame shifts and differences in immunodominance of specific regions among the antisera.…”

Development of vaccines for prevention of botulism

“…The efforts in the laboratory of Atassi and coworkers have focused on the development of a synthetic vaccine against BoNT intoxication [42,43]. The investigators synthesized a series of overlapping 19-mer peptides that spanned the entire H C region of BoNT serotype A (BoNT/ A).…”