Development of an H9N2 vaccine strain with enhanced neuraminidase immunogenicity via dual hemagglutinin and neuraminidase modification.

Abstract

IMPORTANCE: The Y439 lineage 01310 E20 H9N2 vaccine strain currently used in South Korea has undergone extensive egg adaptation, resulting in substantial changes, including an 18-amino acid neuraminidase (NA) stalk deletion. Additionally, both early and late 01310 passages inherently harbor an N-glycan at hemagglutinin (HA) residue 158-160 (HA158) that may interfere with virus-specific antibodies. OBJECTIVE: We aimed to develop a high-yield vaccine strain without egg passaging and to overcome the limitations of the conventional vaccine strain that may compromise immunogenicity. METHODS: We introduced a genetically modified 01310 PB2 gene (310-MVV: I66M, I109V, I133V) to increase replication in embryonated eggs and removed the N-glycan at HA158 and restored the NA stalk to improve immunogenicity. The resulting strain was assessed for egg replication and immunogenicity in chickens. RESULTS: The resulting vaccine strain (310-SNS-193D-MVV) grew efficiently in embryonated eggs without repeated passaging. As restoring the NA stalk alone was insufficient to enhance NA-specific immunity, simultaneously removing the N-glycan at HA158 markedly increased NA-specific antibody responses and neutralizing antibody titers across multiple H9N2 lineages. Additionally, incorporating 310-SNS-193D-MVV into a bivalent formulation with a Y280 lineage strain conferred broader coverage without evidence of immune interference. CONCLUSIONS AND RELEVANCE: These findings underscore how PB2, HA, and NA targeted genetic modifications can improve H9N2 vaccine productivity and immunogenicity. These strategies are not limited to our H9N2 strain and can be applied to other low propagating or NA stalk-deleted virus strains.