Identifying the principal protective antigenic determinants of type A botulinum neurotoxin.
The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned. Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc. However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.
Bavari
S
S
Department of Cell Biology and Biochemistry, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702-5011, USA.
Pless
D D
DD
Torres
E R
ER
Lebeda
F J
FJ
Olson
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001428:Bacterial Vaccines / Q000276:immunology*; D019274:Botulinum Toxins, Type A / Q000276:immunology*; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001428:Bacterial Vaccines; D000939:Epitopes; D010446:Peptide Fragments; D019274:Botulinum Toxins, Type A