Generation and selection of novel human-neutralizing single chain variable fragments against rabies virus from immunized phage display libraries

Rabies remains a deadly infectious disease in tropical areas caused by rabies virus (RABV). Currently, there are limitations in the production and quality control of polyclonal antibodies for postexposure prophylaxis (PEP) for severe patients. Engineered therapeutic monoclonal antibodies (MAbs) are effective therapies against various infectious agents; therefore, we aimed to develop human MAbs against RABV on a single-chain variable fragment (scFv) platform. Immunized scFv libraries were constructed from human peripheral blood mononuclear cells via the pMOD1 phage display system. Diverse VHVLκ and VHVLλ phage-displayed libraries were successfully produced with 2x10 7 and 5.4x10 6 clones, respectively. The high affinity and binding specificity of scFvs targeting the RABV glycoprotein were obtained through biopanning and ELISA. Six unique scFv clones presented diverse complementary determining region (CDR) sequences. Interestingly, HuRABVscFv1 and HuRABVscFv2 exhibited rapid fluorescent foci inhibition test (RFFIT)-neutralizing titers above the 0.5 IU/ml protective level. Finally, in silico molecular docking demonstrated that the CDRs of two neutralizing human clones interact mainly with antigenic sites III (HuRABVscFv1) and II (HuRABVscFv2) on the RABV glycoprotein. These scFvs may be candidates for MAb production against RABV to replace traditional PEP in Thailand. • We successfully generated immunized-human scFv libraries with 10 6 –10 7 clones via the pMOD1 phage display system. • Six distinct scFv clones with various CDR sequences that specifically bind to the RABV glycoprotein were identified. • HuRABVscFv1 and HuRABVscFv2 demonstrated promising neutralizing activities. • In silico molecular docking revealed that these neutralizing clones interact with different binding sites on the RABV glycoprotein, indicating their potential as MAb candidates against RABV infection for postexposure prophylaxis.