Generation and screening of efficient neutralizing single domain antibodies (VHHs) against the critical functional...

Since anthrax is an acute infectious disease, detection and neutralization of the toxins of pathogenic Bacillus anthracis are of great importance. The critical role of protective antigen (PA) component of tripartite anthrax toxin in toxin entry into the host cell cytosol provided a great deal of effort to generate monoclonal antibodies against this constitute. Regarding the importance of anthrax detection/neutralization and unique physicochemical and pharmacological features of VHHs as single domain antibodies, the present study aimed to generate VHHs against the receptor binding domain of PA, termed PAD4. After camel immunization, a gene repertoire of VHH fragments with a diversity of 4.7×108 clones was produced, followed by constructing a VHH phage display library. A stringent successive biopanning was then carried out to isolate the phages displaying high affinity VHHs against PAD4.Polyclonal and monoclonal Enzyme-linked immunosorbent assay (ELISA) verified binding specificity of phages to the target protein. Modeling of VHHs together with the docking simulation studies, illustrated the binding site of antibodies on antigen. Docking analysis revealed that all selected VHHs potently cover the key functional residues of PAD4. Since the selected VHHs could cover and block the receptor binding loops of PA, they could be proposed as hopeful anti-Anthrax candidates.