Characterization of a monoclonal antibody recognizing a conserved linear neutralizing epitope of pseudorabies virus...

Pseudorabies virus (PRV), an α-herpesvirus, infects a variety of mammals. It is mainly prevalent in domestic pigs but can also infect humans, posing significant public health risks. The glycoprotein B (gB), a crucial structural protein involved in viral entry and immune recognition, is a key target for neutralizing antibodies of PRV. This study expressed gB glycoprotein to immunise mice using eukaryotic and prokaryotic systems. The hybridoma cell line secreting anti-gB antibodies was analysed, and the neutralising epitope targeted by this monoclonal antibody was identified. Six anti-PRV monoclonal antibodies were generated via the cell fusion technique. The characterization of mAbs revealed that 4A10 exhibited an IFA titer of 1:3200 and neutralization titer of 1:2⁴ with an antibody affinity of 3.4 × 10⁸ L/mol. The extracellular region of gB was divided into seven segments. The reaction of seven truncated proteins with six mAbs was identified by ELISA, with gB6 identified as the immunodominant epitope. Further truncation revealed that the peptide ⁶¹⁵RVPGERGTC⁶²³ was identified by 4A10 and as a B-cell linear epitope, which was localized within the loop domains of the gB carboxy-terminal (C-Terminal). Homology modeling and molecular docking technology revealed that 4A10 binds to ⁶¹⁵RVPGERGTC⁶²³ through its heavy chain CDR3 variable region. Antigenic epitope mapping revealed that epitope ⁶¹⁵RVPGERGTC⁶²³ is highly conserved among different PRV strains. This study identifies a highly conserved linear neutralizing epitope on PRV gB, providing a molecular basis for improving PRV diagnostic methods and optimizing prevention and control strategies. Pseudorabies virus (PRV) causes substantial economic losses in the swine industry worldwide. Glycoprotein B (gB) is a major target for neutralizing antibodies and vaccine design. In this study, we prepared a neutralizing monoclonal antibody (mAb) against PRV gB and identified its key linear neutralizing epitope. The epitope is highly conserved among prevalent PRV strains, highlighting its critical role in viral antigenicity. This work clarifies the molecular basis of PRV neutralization and identifies a conserved epitope, which upon further validation could serve as a diagnostic candidate and inform antiviral research.