Secretory expression of tetrameric neuraminidase enhanced by signal peptide optimization elicits robust immunity against influenza infection in mice

Neuraminidase (NA), a key surface glycoprotein of influenza viruses, represents a more conserved antigenic target than hemagglutinin (HA) and has attracted interest as a potential target for vaccines with broader protective capacity. Nevertheless, the inefficient secretory production of tetrameric NA that maintain tetramer-like structural features remains challenging.Here, we developed an optimized signal peptide, opt-sp, by introducing valine and asparagine residues at the N-terminus of the Gaussia luciferase signal peptide (Gluc-sp) and incorporating a measles virus phosphoprotein-derived tetramerization domain. In a systematic comparison with three commonly used signal peptides, including tissue plasminogen activator (tPA), immunoglobulin G signal peptide (IgG-sp), and Gluc-sp, opt-sp was associated with increased relative secretion of NA protein under the tested expression conditions. Native PAGE analysis further suggested the presence of tetramer-like NA species. In mice，a DNA vaccine encoding opt-sp-fused NA elicited substantially stronger NA-specific humoral and cellular immune responses than constructs bearing conventional signal peptides and provided improved protection against homologous A/California/04/2009 (H1N1) challenge. These findings identify opt-sp as a promising signal peptide candidate for improving recombinant NA secretion and support further evaluation of this strategy for NA-based influenza vaccine development, including future studies of heterologous protection.