Development of novel canine phage display-derived neutralizing monoclonal antibody fragments against rabies virus from immunized dogs

Animal rabies is a potentially fatal infectious disease in mammals, especially dogs. Currently, the number of rabies cases in pet dogs is increasing in several regions of Thailand. However, no passive postexposure prophylaxis (PEP) has been developed to combat rabies infection in animals. As monoclonal antibodies (MAbs) are promising biological therapies for postinfection, we developed a canine-neutralizing MAb against rabies virus (RABV) via the single-chain variable fragment (scFv) platform. Immunized phage-displaying scFv libraries were constructed from PBMCs via the pComb3XSS system. Diverse canine VHVLκ and VHVLλ libraries containing 2.4 × 10⁸ and 1.3 × 10⁶ clones, respectively, were constructed. Five unique clones that show binding affinity with the RABV glycoprotein were then selected, of which K9RABVscFv1 and K9RABVscFv16 showed rapid fluorescent foci inhibition test (RFFIT) neutralizing titers above the human protective level of 0.5 IU/ml. Finally, in silico docking predictions revealed that the residues on the CDRs of these neutralizing clones interact mainly with similar antigenic sites II and III on the RABV glycoprotein. These candidates may be used to develop complete anti-RABV MAbs as a novel PEP protocol in pet dogs and other animals.