Wen-Chun Liu, Chia-Ying Lin, Yung-Ta Tsou, Jia-Tsrong Jan, Suh-Chin Wu

Neuraminidase (NA), an influenza virus envelope glycoprotein, removes sialic acid from receptors for virus release from infected cells. For this study we used a baculovirus-insect cell expression system to construct and purify H5N1-rNA (A/Vietnam/1203/2004) and pH1N1-rNA (A/Texas/05/2009) proteins. BALB/c mice immunized with these proteins had high titers of NA-specific IgG and NA-inhibiting (NI) antibodies against H5N1, pH1N1, H3N2 and H7N9 viruses. H5N1-rNA immunization resulted in higher quantities of NA-specific antibody-secreting B cells in spleen against H5N1 and heterologous pH1N1 viruses. H5N1-rNA and pH1N1-rNA immunizations both provided complete protection against homologous virus challenges, with H5N1-rNA immunization providing better protection against pH1N1 virus challenges. Cross-reactive NI antibodies were further dissected via pH1N1-rNA protein immunizations with I149V, N344Y and I365T/S366N NA mutations. The I365T/S366N mutation of pH1N1-rNA enhanced cross-reactive NI antibodies against H5N1, H3N2 and H7N9 viruses. It is our hope that these findings provide useful information for the development of a NA-based universal influenza vaccine. Neuraminidase (NA) is an influenza virus enzymatic protein that cleaves sialic acid linkages on infected cell surfaces, thus facilitating viral release and contributing to viral transmission and mucus infection. In currently available inactivated or live-attenuated influenza vaccines based on the antigenic content of hemagglutinin proteins, vaccine efficacy can be partly contributed through NA-elicited immune responses. We investigated the NA immunity of different recombinant NA (rNA) proteins associated with pH1N1 and H5N1 viruses. Our results indicate that H5N1-rNA immunization induced more potent cross-protective immunity than pH1N1-rNA immunization, and three residues at I149V, I365T and S366N near the NA enzyme active site(s) are linked to enhanced cross-reactive NA-inhibiting antibodies against heterologous and heterosubtypic influenza A viruses. These findings provide useful information for the development of a NA-based universal influenza vaccine.