Identification of a novel conserved B-cell epitope in the glycoprotein of rabies virus.

Abstract

Rabies virus (RABV), a single-stranded RNA virus, invades the central nervous system and causes acute, zoonotic diseases worldwide. The glycoprotein (G) of RABV is the only surface antigen responsible for inducing potent neutralizing antibodies, making it a key target for medical countermeasures. In this study, two anti-G protein monoclonal antibodies (mAbs) were prepared using hybridoma cell technology, with their high specificity and robust reactivity validated by indirect enzyme-linked immunosorbent assay (i-ELISA), indirect immunofluorescence assay (IFA) and Western blot. Through systematic epitope mapping using a series of truncated G protein fragments, two mAbs (1G2 and 4E5) were found to recognize a minimal linear epitope, AEDFVEVHLP (amino acids 412-421) through Dot-ELISA and i-ELISA. Alanine-scanning mutagenesis further defined the core binding residues within this epitope as ⁴¹²Ala-Glu-Asp-Phe-Val⁴¹⁶, ⁴¹⁸Val, ⁴²⁰Leu, and ⁴²¹Pro. Bioinformatic analysis with MEGA software revealed highly conserved with conservative substitutions of this epitope across diverse RABV of genotype 1. Homology modeling with SWISS-MODEL localized the epitope "⁴¹²AEDFVEVHLP⁴²¹" to a solvent-exposed α-helical conformation on the G protein surface. And this epitope is located in the extracellular C-terminal region, Prior studies have demonstrated that this region plays a role in stabilizing the trimerization of the rabies virus glycoprotein (RVG). In conclusion, our study identifies a novel, highly conserved with conservative substitutions linear B-cell epitope on the RABV glycoprotein, providing a precise molecular target for the rational design of next-generation epitope-based vaccines and specific diagnostic tools.