A dual strategy - in vitro and in silico - to evaluate human antitetanus mAbs addressing their potential protective action on TeNT endocytosis in primary rat neuronal cells

Abstract

Tetanus disease is caused by the spore-forming bacteria Clostridium tetani. Its development starts with wounds or mucous layers of spore contact in water, soil, human, and animal feces. It is prevented by vaccination, but the lack of booster shots throughout life and age-decreased immune surveillance by immunosenescence enforces prophylactic treatment in case of accidents. The disease incident and lethality present a high index in underdeveloped countries. In cases of infection, administering antitetanus antibodies is preconized, usually derived from immunized horses or humans. Heterologous sera origin and the total protein content represent risks such as serum sickness and serum sickness-like reactions. Human sera can carry unknown viruses. The search for human monoclonal antibodies (mAbs) against TeNT (tetanus toxin) has increased in the last few years, although none has been approved. From a panel of previously identified human mAbs derived from B-cell sorting, we selected two nonrelated mAbs that bind to HCR/T (fragment C) of TeNT, showing disruption of its interaction with the cellular receptor ganglioside GT1b. To gain more insight into the TeNT inhibition effectiveness, we present results based on cellular assays and molecular docking tools. We describe TeNT internalization in neurons derived from the neonatal rat spinal cord. Adding the single mAbs prevented TeNT internalization higher than 50% under a specific experimental condition. The data validation was performed by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the Synaptic Vesicle Glycoprotein (SV2) mediator role in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes of the mAbs showed the binding of both antibodies to the heavy chain of TeNT, specifically to the HCR/T domain. A higher incidence was found between N1153 and W1289 when evaluating candidate residues for conformational epitope. AUTHOR SUMMARYTetanus neurotoxin, produced by the bacteria C. tetani, is one of the most potent toxins. The ubiquitous presence of bacteria and spores in soil, water, and human and animal feces makes their elimination impossible, and accidents occur in contact with wounds. People who work in agricultural or animal production are more susceptible to infection. In recent years, there has been an increase in groups developing human monoclonal antibodies for specific immunotherapy. Our group screened a panel of monoclonal antibodies in search of the best neutralizing compositions. This work reports convergent in vitro and in silico results on two antibodies that bind to the toxin domain, which interacts with neuronal cells and initiates the disease pathway. We show the hydrophobic interactions and hydrogen bonds that make contact between each antibody and the toxin, the entry of the toxin into the rat primary spinal cord cells, its interaction with synaptic vesicle II, and how the antibodies reduce the entry of the toxin into the cells, pointing to their neutralizing potential.