Monoclonal Antibodies Specific for the Hemagglutinin-Neuraminidase Protein Define Neutralizing Epitopes Specific for Newcastle Disease Virus Genotype 2.VII from Egypt

Background: Newcastle disease is a devastating disease in poultry caused by Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analysis reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Heamagglutinin-Neuraminidase (HN) spike protein, we were interested to established genotype-specific monoclonal antibodies (MAbs). Methods: An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induced antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilize Concanavalin A (ConA-ELISA) coupled Glycoproteins that was proven to present conformation-dependent epitopes. Results: Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI-activity. One MAb recognized an epitope only present in the homologue virus while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. Conclusions: These results point to the concurrent presence of variable and conserved epitopes within the HN-molecule of NDV. The described protocol should help to generate MAbs to a variety of NDV strains and enable in depth analysis of the antigenic profiles of different genotypes.