A novel monoclonal antibody targeting the hemagglutinin-neuraminidase of peste des petits ruminants virus maintains neutralizing activity by blocking viral adsorption and receptor interaction

Peste des petits ruminants (PPR) is a highly contagious viral disease that primarily affects sheep and goats, posing a significant threat to small ruminant livestock production. The protective humoral immunity against Peste des petits ruminants virus (PPRV) is mainly mediated by the Hemagglutinin-Neuraminidase (HN) glycoprotein. To elucidate the functional antigenic determinants of the PPRV HN protein, we developed a new monoclonal antibody (mAb), HN^1D4-4D9. Epitope mapping analysis revealed that HN^1D4-4D9 recognized a novel, conserved epitope (³⁸⁰ECLVEACK³⁸⁷), including a linear epitope (³⁸¹CLVEACK³⁸⁷), and a conformational epitope (³⁸⁰ECLVEA³⁸⁵) across multiple PPRV genotypes. Alanine-scanning mutagenesis identified key residues within the conserved linear and conformational epitopes of the PPRV HN protein. Surface plasmon resonance analysis demonstrated the high-affinity binding of the identified HN epitope peptide to HN^1D4-4D9. Using virus pre-treatment and virus-antibody mixture neutralizing assays, HN^1D4-4D9 exhibited potent neutralizing activity in vitro, whereas the post-attachment assay showed that HN^1D4-4D9 was ineffective after virus binding to cells, underscoring that the antibody acts at the viral adsorption stage. Mechanistically, quantitative Real-Time PCR and competitive co-immunoprecipitation assays revealed that HN^1D4-4D9 blocks viral adsorption by preventing HN interaction with the receptors SLAM and Nectin-4, rather than by interfering with the fusion protein. Further biochemical assays revealed that several amino acid residues within the novel epitope are critical for the interaction with both receptors. These findings identify a critical neutralizing epitope on PPRV HN, provide mechanistic insights into antibody-mediated viral inhibition, and support the potential application of HN^1D4-4D9 as a candidate for epitope-based vaccine optimization, development of entry-targeted Antiviral drugs, and diagnostic and prophylactic strategies for controlling PPR.IMPORTANCEPeste des petits ruminants virus (PPRV) is a highly contagious morbillivirus that causes severe disease in sheep and goats, posing a significant threat to global small ruminant production. Viral entry is initiated by the Hemagglutinin-Neuraminidase (HN) glycoprotein through its interaction with host receptors. In this study, we identified a novel monoclonal antibody, HN^1D4-4D9, that targets a highly conserved neutralizing epitope (³⁸⁰ECLVEACK³⁸⁷) on the PPRV HN protein and exhibits potent in vitro Antiviral activity. Mechanistically, HN^1D4-4D9 blocks viral adsorption by preventing HN engagement with the critical cellular receptors SLAM and Nectin-4, thereby inhibiting Infection at the earliest stage of the viral life cycle. These findings define a key functional neutralizing site on HN and provide essential insights into the molecular basis of PPRV entry and antibody-mediated inhibition. The identified epitope represents a promising target for next-generation epitope-based vaccines and entry-targeted Antiviral therapeutics. In addition, HN^1D4-4D9 may have potential applications in passive immunoprophylaxis, diagnostics, and studies of PPRV pathogenesis.