Development and Epitope Characterization of Monoclonal Antibodies Targeting the Rabies Virus P Protein

Rabies is a fatal zoonotic disease caused by rabies virus (RABV), resulting in approximately 59,000 deaths annually worldwide and posing a serious threat to public health. The RABV phosphoprotein (P protein) plays crucial roles in viral replication, transcription, and immune antagonism; however, its immunogenic properties have not been fully characterized. In this study, the RABV P protein was expressed in an Escherichia coli expression system and used to immunize mice, resulting in the generation of six P protein-specific monoclonal antibodies (mAbs). Using an overlapping peptide-based truncation strategy, two linear B-cell epitopes were identified: ⁵²DMKRLHLDDEKSSNL⁶⁶ and ¹⁷⁷VAPGPPALEWSATNE¹⁹¹. Alanine-scanning mutagenesis revealed that residues D52, M53, R55, L56, and L58 were critical for the recognition of epitope ⁵²DMKRLHLDDEKSSNL⁶⁶ by mAbs 2D2 and 18G7. Residues G180, P181, and W186 were essential for recognition of epitope ¹⁷⁷VAPGPPALEWSATNE¹⁹¹ by mAbs 3D7, 15D8, 16D3, and 16C6. Notably, although some amino acid residues within epitopes P1-5 and P4-2 exhibited high variability among representative RABV strains, the critical residues recognized by these monoclonal antibodies were highly conserved. These findings provide new insights into the antigenic structure of the RABV P protein and may contribute to future studies on its functional characterization, as well as the development of P protein-based diagnostic reagents and subunit vaccines.