Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries.
نویسندگان
چکیده
To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H(C). Twenty-eight unique BoNT/A H(C) binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, respectively, compared to toxin control. scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.
منابع مشابه
تهیه نانو واکسن نوترکیب نوروتوکسین بوتولینوم تیپ E بر پایه کیتوسان و مقایسه ایمنیزایی آن به دو روش تزریقی و خوراکی در موش سوری
Background and Objectives: Botulism syndrome is caused by one of the seven botulinum neurotoxins. The toxins binding domain have immunogenicity effect and can be used as a recombinant vaccine candidate against botulism disease. Due to the low immunogenicity of recombinant protein , the use of an appropriate vehicle for antigen delivery to target cells is inevitable. The purpose of this study ...
متن کاملImmunogenic and Protective Potentials of Recombinant Receptor Binding Domain and a C-Terminal Fragment of Clostridium botulinum Neurotoxin Type E
Clostridium Botulinum Type E neurotoxin heavy chain consists of two domains: the translocation domain asthe N-terminal half and the binding domain as the Cterminal half (Hc). One effective way to neutralize botulinum neurotoxin is to inhibit binding of this toxin to neuromuscular synapses with antibodies against binding domain. Two synthetic genes, coding for Hc (the full length binding d...
متن کاملRecombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo
Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most ...
متن کاملCloning, Expression and Purification of Clostridium botulinum Neurotoxin Type E Binding Domain
متن کامل
Comparative Study of Immunological and Structural Properties of Two Recombinant Vaccine Candidates against Botulinum Neurotoxin Type E
Background: Recently, botulinum neurotoxin (BoNT)-derived recombinant proteins have been suggested as potential botulism vaccines. Here, with concentrating on BoNT type E (BoNT/E), we studied two of these binding domain-based recombinant proteins: a multivalent chimer protein, which is composed of BoNT serotypes A, B and E binding subdomains, and a monovalent recombinant protein, which contains...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Infection and immunity
دوره 65 9 شماره
صفحات -
تاریخ انتشار 1997